http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Patricia&year=&month=
2007.igem.org - User contributions [en]
2024-03-29T06:26:05Z
From 2007.igem.org
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http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2008-02-15T11:27:34Z
<p>Patricia: /* Contact */</p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
<br />
<br />
=====Education=====<br />
I am currently completing my third year of a Bachelor of Science degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me, iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse all our undertakings in the lab.<br />
<br />
=====Interests outside of science include:=====<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2007-10-25T05:40:51Z
<p>Patricia: /* Education */</p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
=====Contact=====<br />
pat@illing.com.au<br />
<br />
=====Education=====<br />
I am currently completing my third year of a Bachelor of Science degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me, iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse all our undertakings in the lab.<br />
<br />
=====Interests outside of science include:=====<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_used
Melbourne/Parts used
2007-10-25T05:39:29Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: 25/10/07<br />
{| border="1"<br />
|+ Parts Used by Melbourne [[Melbourne/parts used graphical| <GRAPHICAL>]]<br />
! Label !! Registry Part Number !! Part Name !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status<br />
|-<br />
! [[Melbourne/BBa_B0014|P1 1G]]<br />
|[http://partsregistry.org/Part:BBa_B0014 BBa_B0014] || Double Terminator || 95 || pSB1AK3 || 3189 || 3284 || Amp, Kan || Glycerol stocks in -80<br />
|-<br />
! [[Melbourne/BBa_B0034|P1 3O]]<br />
| [http://partsregistry.org/Part:BBa_B0034 BBa_B0034] || RBS || 12 || pSB1A2 || 2079 || 2091 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_C0051|P1 5G]]<br />
| [http://partsregistry.org/Part:BBa_C0051 BBa_C0051] || c1 represser protein || 750 || pSB1A2 || 2079 || 2892 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0040|P1 5H]]<br />
| [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] || GFP || 720 || pSB1A2 || 2079 || 2799 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0430|P1 11A]]<br />
| [http://partsregistry.org/Part:BBa_E0430 BBa_E0430] || EYFP(RBS+,LVA-,term) || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0084|P1 11H]]<br />
| [http://partsregistry.org/Part:BBa_R0084 BBa_R0084] || OmpR Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0082|P1 15P]]<br />
| [http://partsregistry.org/Part:BBa_R0082 BBa_R0082] || OmpR+ Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0840|P1 16E]]<br />
| [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] || RBS,GFP,term || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0083|P1 17H]]<br />
| [http://partsregistry.org/Part:BBa_R0083 BBa_R0083] || OmpR+ Promoter(truncated version of R0082 || 79 || pSB1A2 || 2079 || 2157 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_B0010|P2 3P]]<br />
| [http://partsregistry.org/Part:BBa_B0010 BBa_B0010] || Single Terminator || 80 || pSB1A2 || 2079 || 2159 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0033|P2 9E]]<br />
| [http://partsregistry.org/Part:BBa_E0033 BBa_E0033] || LacZ alpha || 353 || pSB2K3 || 4425 || 4778 || Kan || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_Q04510|P2 13K]]<br />
| [http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] || c1 inverter || 987 || pSB2K3 || 4425 || 5412 || Kan || Transformed<br />
|-<br />
! [[Melbourne/BBa_E0241|P2 15L]]<br />
| [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] || PoPs -> GFP || 795 || pSB1A2 || 2079 || 2874 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15008|P2 21A]]<br />
| [http://partsregistry.org/Part:BBa_I15008 BBa_I15008] || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AC|P2 21B]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR/ChlR || 675 || pSB1AC3 || 3055 || 3730 || A/C || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15009|P2 21C]]<br />
| [http://partsregistry.org/Part:BBa_I15009 BBa_I15009] || PcyA || 750 || pSB2K3 || 4425 || 5175 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AK|P2 23N]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR/KanR || 675 || pSB1AK3 || 3189 || 3864 || Amp.Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_A|P3 20I]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR || 675 || pSB1A3 || 2157 || 2832 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61035|P4 8J]]<br />
| [http://partsregistry.org/Part:BBa_J61035 BBa_J61035] || EcoRI-GenR-XbaI || 3551 || J61035 || 3551 || 3551 || Amp, Gen || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15010|I15010]]<br />
| [http://partsregistry.org/Part:BBa_I15010 BBa_I15010] || cph8(cph1/Envz fusion || 2238 || pSB2K3 || 4425 || 6663 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/pJS010|pJS010]]<br />
| - || SopII-Htr || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/Fusion|Fusion]]<br />
| - || NpHtrII fusion || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/CP919| CP919]]<br />
| - || EnvZ- strain || || || || || KanR || Glycerol stock in -80/comp. cells<br />
|-<br />
! [[Melbourne/M30109| M30109]]<br />
| [http://partsregistry.org/Part:BBa_M30109 BBa_M30109] || Red red responsive system, dual regulation ||4333||??||??||??|| AmpR + CmR || Discontinued<br />
|-<br />
! [[Melbourne/BBa_R0040|P17O]]<br />
| [http://partsregistry.org/Part:BBa_R0040 BBa_R0040] || tetR negative promoter ||54 ||pSB1A2|| 2079||2133 || AmpR || Transformed<br />
|-<br />
! [[Melbourne/BBa_B0015| P11I]]<br />
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] || Double Terminator ||129||pSB1AK3|| 3189 ||3318|| AmpR + KanR ||Transformed<br />
|-<br />
! [[Melbourne/BBa_J61034| P48F]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || IPTG sensory device ||1417||pSB2K3 || 4425 ||5842|| KanR || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61034| P48H]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || IPTG sensory device ||1417||BBa_J61002 || 3016 ||4433|| AmpR || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61002| P31E]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || Ptet sensory device ||78||pSB2A3 || 2934||3002|| AmpR || Glycerol stock in -80<br />
|-<br />
<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_A
Melbourne/BBa P1010 A
2007-10-25T05:38:50Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:Death plasmid=<br />
Last updated: 25/10/07<br />
<br />
==Physical DNA==<br />
*Registry: BBa_P1010<br />
*Kit location: '''P3 20I'''<br />
*Resistance: Amp<br />
*Part Size:675<br />
*Plasmid Vector: pSB1A3<br />
*Vector Size: 2157<br />
*Total Size: 2832<br />
<br />
==Status==<br />
*Labelling: '''P3 20I'''<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: 31/7<br />
***Location: -20<br />
**Confirmed: yes<br />
**Glycerol Stock: 31/7<br />
***Location:-80<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_AC
Melbourne/BBa P1010 AC
2007-10-25T05:36:33Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:BBa_P1010 A/C=<br />
Last updated:25/10/07<br />
<br />
==Physical DNA==<br />
*Registry: BBa_P1010<br />
*Kit location: '''P2 21B'''<br />
*Resistance: Amp, Chl<br />
*Part Size: 675<br />
*Plasmid Vector: pSB1AC3<br />
*Vector Size: 3055<br />
*Total size: 3730<br />
<br />
==Status==<br />
*Labelling:'''P2 21B'''<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: 31/7<br />
***Location: -20<br />
**Confirmed: yes<br />
**Glycerol Stock: 31/7<br />
***Location:-80<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_AC
Melbourne/BBa P1010 AC
2007-10-25T05:35:48Z
<p>Patricia: /* Status */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:BBa_P1010 A/C=<br />
Last updated:25/10/07<br />
P2 21B BBa_P1010 Death Plasmid AmpR/ChlR 675 pSB1AC3 3055 3730 A/C Glycerol stock in -80 <br />
==Physical DNA==<br />
*Registry: BBa_P1010<br />
*Kit location: '''P2 21B'''<br />
*Resistance: Amp, Chl<br />
*Part Size: 675<br />
*Plasmid Vector: pSB1AC3<br />
*Vector Size: 3055<br />
*Total size: 3730<br />
<br />
==Status==<br />
*Labelling:'''P2 21B'''<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: 31/7<br />
***Location: -20<br />
**Confirmed: yes<br />
**Glycerol Stock: 31/7<br />
***Location:-80<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_AC
Melbourne/BBa P1010 AC
2007-10-25T05:35:29Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:BBa_P1010 A/C=<br />
Last updated:25/10/07<br />
P2 21B BBa_P1010 Death Plasmid AmpR/ChlR 675 pSB1AC3 3055 3730 A/C Glycerol stock in -80 <br />
==Physical DNA==<br />
*Registry: BBa_P1010<br />
*Kit location: '''P2 21B'''<br />
*Resistance: Amp, Chl<br />
*Part Size: 675<br />
*Plasmid Vector: pSB1AC3<br />
*Vector Size: 3055<br />
*Total size: 3730<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: 31/7<br />
***Location: -20<br />
**Confirmed: yes<br />
**Glycerol Stock: 31/7<br />
***Location:-80<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_Q04510
Melbourne/BBa Q04510
2007-10-25T05:29:21Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:c1 inverter=<br />
Last updated: 25/10/07<br />
==Physical DNA==<br />
*Registry: BBa_Q04510<br />
*Kit location: '''P2 13K'''<br />
*Resistance: Kan<br />
*Part Size: 987<br />
*Plasmid Vector: pSB2K3<br />
*Vector Size: 4425<br />
*Total Size: 5412<br />
<br />
==Status==<br />
*Labelling: '''P2 13K'''<br />
*Resuspended: Yes<br />
**Location: -20 freezer<br />
*Transformed: 12/8<br />
**Location: Cold Room<br />
**Miniprepped: 14/8<br />
***Location: -20 freezere<br />
***Confirmed: 14/8<br />
**Glycerol Stock: 14/8 <br />
***Location:-80 freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_E0033
Melbourne/BBa E0033
2007-10-25T05:26:52Z
<p>Patricia: /* Status */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:25/10/07<br />
==Physical DNA==<br />
*Registry: BBa_E0033<br />
*Kit location: ''P2 9E''<br />
*Resistance: Kan<br />
*Part Size: 353<br />
*Plasmid Vector: pSB2K3<br />
*Vector Size: 4425<br />
*Total size: 4778<br />
<br />
==Status==<br />
*Labelling: "P2 9E"<br />
*Resuspended: 1/8<br />
**Location: -20 freezer<br />
*Transformed: 1/8<br />
**Location: cold room<br />
**Miniprepped: 6/8<br />
***Location: -20 freezer<br />
**Confirmed: 6/8<br />
**Glycerol Stock: 6/8<br />
***Location: -80 freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_E0033
Melbourne/BBa E0033
2007-10-25T05:26:29Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:25/10/07<br />
==Physical DNA==<br />
*Registry: BBa_E0033<br />
*Kit location: ''P2 9E''<br />
*Resistance: Kan<br />
*Part Size: 353<br />
*Plasmid Vector: pSB2K3<br />
*Vector Size: 4425<br />
*Total size: 4778<br />
<br />
==Status==<br />
*Labelling: ''P2 9E''<br />
*Resuspended: 1/8<br />
**Location: -20 freezer<br />
*Transformed: 1/8<br />
**Location: cold room<br />
**Miniprepped: 6/8<br />
***Location: -20 freezer<br />
**Confirmed: 6/8<br />
**Glycerol Stock: 6/8<br />
***Location: -80 freezer<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_R0083
Melbourne/BBa R0083
2007-10-25T05:22:02Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:Truncated OmpR+ promoter - truncated version of R0082=<br />
Last updated: 25/10/2007<br />
<br />
==Physical DNA==<br />
*Registry: BBa_R0083<br />
*Kit location: '''P1 17H''' <br />
*Resistance: Amp<br />
*Part Size: 79<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
*Total Size: 2158<br />
<br />
==Status==<br />
*Labelling: '''P1 17H'''<br />
*Resuspended: yes<br />
**Location: -20 freezer box 1<br />
*Transformed: 8/7<br />
**Location: cold room<br />
**Miniprepped: 10/7<br />
***Location: -20 freezer miniprep box<br />
**Confirmed: yes<br />
**Glycerol Stock: labelled 10/7, performes 11/7<br />
***Location: -80 freezer<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/PstI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_E0840
Melbourne/BBa E0840
2007-10-25T05:20:58Z
<p>Patricia: /* Name: RBS, GFP, ter */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name: RBS, GFP, ter =<br />
Last updated: 25/10/2007<br />
<br />
==Physical DNA==<br />
*Registry: BBa_E0840<br />
*Kit location: '''P1 16E'''<br />
*Resistance: Amp<br />
*Part Size: 878<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
*Total size: 2957<br />
<br />
==Status==<br />
*Labelling: '''P1 16E'''<br />
*Resuspended: yes<br />
**Location: -20 freezer in box 1<br />
*Transformed: 8/7<br />
**Location: cold room<br />
**Miniprepped: 10/7<br />
***Location: -20 freezer miniprep box<br />
**Confirmed: yes<br />
**Glycerol Stock: labelled 10/7, performed 11/7<br />
***Location:-80 freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/PstI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_E0840
Melbourne/BBa E0840
2007-10-25T05:20:43Z
<p>Patricia: /* Status */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name: RBS, GFP, ter =<br />
Last updated: 10/07/2007<br />
<br />
==Physical DNA==<br />
*Registry: BBa_E0840<br />
*Kit location: '''P1 16E'''<br />
*Resistance: Amp<br />
*Part Size: 878<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
*Total size: 2957<br />
<br />
==Status==<br />
*Labelling: '''P1 16E'''<br />
*Resuspended: yes<br />
**Location: -20 freezer in box 1<br />
*Transformed: 8/7<br />
**Location: cold room<br />
**Miniprepped: 10/7<br />
***Location: -20 freezer miniprep box<br />
**Confirmed: yes<br />
**Glycerol Stock: labelled 10/7, performed 11/7<br />
***Location:-80 freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/PstI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_R0082
Melbourne/BBa R0082
2007-10-25T05:19:23Z
<p>Patricia: /* Name:OmpR+ promoter */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:OmpR+ promoter=<br />
Last updated: 25/10/2007<br />
<br />
==Physical DNA==<br />
*Registry: BBa_R0082<br />
*Kit location: '''P1 15P'''<br />
*Resistance: Amp<br />
*Part Size: 108<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
<br />
==Status==<br />
*Labelling: '''P1 15P<br />
*Resuspended: yes<br />
**Location: -20 freezer box 1<br />
*Transformed: yes<br />
**Location: cold room<br />
**Miniprepped: 10/7<br />
***Location: -20 freezer miniprep box<br />
**Confirmed: yes<br />
**Glycerol Stock: labelled 10/7, performed 11/7<br />
***Location:-80 degree freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/PstI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_R0082
Melbourne/BBa R0082
2007-10-25T05:19:04Z
<p>Patricia: /* Status */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:OmpR+ promoter=<br />
Last updated: 10/07/2007<br />
<br />
==Physical DNA==<br />
*Registry: BBa_R0082<br />
*Kit location: '''P1 15P'''<br />
*Resistance: Amp<br />
*Part Size: 108<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
<br />
==Status==<br />
*Labelling: '''P1 15P<br />
*Resuspended: yes<br />
**Location: -20 freezer box 1<br />
*Transformed: yes<br />
**Location: cold room<br />
**Miniprepped: 10/7<br />
***Location: -20 freezer miniprep box<br />
**Confirmed: yes<br />
**Glycerol Stock: labelled 10/7, performed 11/7<br />
***Location:-80 degree freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/PstI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_R0084
Melbourne/BBa R0084
2007-10-25T05:17:48Z
<p>Patricia: /* Status */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:OmpR positive promoter=<br />
<br />
==Physical DNA==<br />
*Registry: BBa_0084<br />
*Kit location: '''P1 11H'''<br />
*Resistance: Amp<br />
*Part Size: 108<br />
*Plasmid Vector: pSB1A2<br />
*Vector Size: 2079<br />
*Total size: 2187<br />
<br />
==Status==<br />
*Labelling: '''P1 11H'''<br />
*Resuspended: Yes<br />
**Location: -20 freezer<br />
*Transformed: Yes<br />
**Location: Cold room<br />
**Miniprepped: 7/7<br />
***Location: -20 freezer<br />
**Confirmed: 7/7<br />
**Glycerol Stock: 7/7<br />
***Location:-80 freezer<br />
<br />
==Expected Digestion Products==<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_J61035
Melbourne/BBa J61035
2007-10-25T05:14:43Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:EcoRI-GenR-XbaI-RBS=<br />
<br />
==Physical DNA==<br />
*Registry: BBa_J61035<br />
*Kit location: '''P4 8J'''<br />
*Resistance: Amp, Gen<br />
*Part Size: 3551<br />
*Plasmid Vector: -<br />
*Vector Size: - <br />
<br />
==Status==<br />
*Labelling: '''P4 8J'''<br />
*Resuspended: Yes<br />
**Location: -20 freezer<br />
*Transformed: yes<br />
**Location: cold room<br />
**Miniprepped: 6/8<br />
***Location: -20 freezer<br />
**Confirmed: 6/8<br />
**Glycerol Stock: 6/8<br />
***Location:-80 freezer<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_used
Melbourne/Parts used
2007-10-25T05:12:12Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: 25/10/07<br />
{| border="1"<br />
|+ Parts Used by Melbourne [[Melbourne/parts used graphical| <GRAPHICAL>]]<br />
! Label !! Registry Part Number !! Part Name !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status<br />
|-<br />
! [[Melbourne/BBa_B0014|P1 1G]]<br />
|[http://partsregistry.org/Part:BBa_B0014 BBa_B0014] || Double Terminator || 95 || pSB1AK3 || 3189 || 3284 || Amp, Kan || Glycerol stocks in -80<br />
|-<br />
! [[Melbourne/BBa_B0034|P1 3O]]<br />
| [http://partsregistry.org/Part:BBa_B0034 BBa_B0034] || RBS || 12 || pSB1A2 || 2079 || 2091 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_C0051|P1 5G]]<br />
| [http://partsregistry.org/Part:BBa_C0051 BBa_C0051] || c1 represser protein || 750 || pSB1A2 || 2079 || 2892 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0040|P1 5H]]<br />
| [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] || GFP || 720 || pSB1A2 || 2079 || 2799 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0430|P1 11A]]<br />
| [http://partsregistry.org/Part:BBa_E0430 BBa_E0430] || EYFP(RBS+,LVA-,term) || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0084|P1 11H]]<br />
| [http://partsregistry.org/Part:BBa_R0084 BBa_R0084] || OmpR Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0082|P1 15P]]<br />
| [http://partsregistry.org/Part:BBa_R0082 BBa_R0082] || OmpR+ Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0840|P1 16E]]<br />
| [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] || RBS,GFP,term || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0083|P1 17H]]<br />
| [http://partsregistry.org/Part:BBa_R0083 BBa_R0083] || OmpR+ Promoter(truncated version of R0082 || 79 || pSB1A2 || 2079 || 2157 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_B0010|P2 3P]]<br />
| [http://partsregistry.org/Part:BBa_B0010 BBa_B0010] || Single Terminator || 80 || pSB1A2 || 2079 || 2159 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0033|P2 9E]]<br />
| [http://partsregistry.org/Part:BBa_E0033 BBa_E0033] || LacZ alpha || 353 || pSB2K3 || 4425 || 4778 || Kan || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_Q04510|P2 13K]]<br />
| [http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] || c1 inverter || 987 || pSB2K3 || 4425 || 5412 || Kan || Transformed<br />
|-<br />
! [[Melbourne/BBa_E0241|P2 15L]]<br />
| [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] || PoPs -> GFP || 795 || pSB1A2 || 2079 || 2874 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15008|P2 21A]]<br />
| [http://partsregistry.org/Part:BBa_I15008 BBa_I15008] || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AC|P2 21B]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR/ChlR || 675 || pSB1AC3 || 3055 || 3730 || A/C || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15009|P2 21C]]<br />
| [http://partsregistry.org/Part:BBa_I15009 BBa_I15009] || PcyA || 750 || pSB2K3 || 4425 || 5175 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AK|P2 23N]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR/KanR || 675 || pSB1AK3 || 3189 || 3864 || Amp.Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_A|P3 20I]]<br />
| [http://partsregistry.org/Part:BBa_P1010 BBa_P1010] || Death Plasmid AmpR || 675 || pSB1A3 || 2157 || 2832 || Kan || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61035|P4 8J]]<br />
| [http://partsregistry.org/Part:BBa_J61035 BBa_J61035] || EcoRI-GenR-XbaI || 3551 || J61035 || 3551 || 3551 || Amp, Gen || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15010|I15010]]<br />
| [http://partsregistry.org/Part:BBa_I15010 BBa_I15010] || cph8(cph1/Envz fusion || 2238 || pSB2K3 || 4425 || 6663 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/pJS010|pJS010]]<br />
| - || SopII-Htr || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/Fusion|Fusion]]<br />
| - || NpHtrII fusion || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/CP919| CP919]]<br />
| - || EnvZ- strain || || || || || KanR || Glycerol stock in -80/comp. cells<br />
|-<br />
! [[Melbourne/M30109| M30109]]<br />
| [http://partsregistry.org/Part:BBa_M30109 BBa_M30109] || Red red responsive system, dual regulation ||4333||??||??||??|| AmpR + CmR || Discontinued<br />
|-<br />
! [[Melbourne/BBa_R0040|P17O]]<br />
| [http://partsregistry.org/Part:BBa_R0040 BBa_R0040] || tetR negative promoter ||54 ||pSB1A2|| 2079||2133 || AmpR || Transformed<br />
|-<br />
! [[Melbourne/BBa_B0015| P11I]]<br />
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] || Double Terminator ||129||pSB1AK3|| 3189 ||3318|| AmpR + KanR ||Transformed<br />
|-<br />
! [[Melbourne/BBa_J61034| P48F]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || IPTG sensory device ||1417||pSB2K3 || 4425 ||5842|| KanR || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61034| P48H]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || IPTG sensory device ||1417||BBa_J61002 || 3016 ||4433|| AmpR || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61002| P31E]]<br />
| [http://partsregistry.org/Part:BBa_J61034 BBa_J61034] || Ptet sensory device ||78||pSB2A3 || 2934||3002|| AmpR || Glycerol stock in -80<br />
|-<br />
<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_17-18
Melbourne/Lab Notebook Weeks 17-18
2007-10-25T05:10:26Z
<p>Patricia: /* Week 18 */</p>
<hr />
<div>[[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne| <team home page>]]<br />
<br />
<br />
==Week 17==<br />
<font size=3><b>14 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>15 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>16 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>17 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>18 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>19 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>20 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
==Week 18==<br />
<font size=3><b>21 Oct 2007 <br />
</b><br />
</font><BR><br />
*Sequenced GAT2 and GPT4 from vr<br />
<br />
<font size=3><b>22 Oct 2007 <br />
</b><br />
</font><BR><br />
*GAT2 and GPT sequence confirmed identities as:<br />
**GAT2 = GenRBS-ComA-ter<br />
**GPt4 = GenRBS-ComP-ter<br />
<br />
<font size=3><b>23 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>24 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>25 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>26 Oct 2007 <br />
</b><br />
</font><BR></div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_13-16
Melbourne/Lab Notebook Weeks 13-16
2007-10-25T05:08:16Z
<p>Patricia: /* Week 16 */</p>
<hr />
<div>[[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne| <team home page>]]<br />
<br />
==Week 13==<br />
<font size=3><b>16 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>17 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] 2 cultures from each of the transformations on 12/9 and 4 cultures from Omp-C1-reporter.<br />
*[[Melbourne/Diagnostic Digest| Digested]] these and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# P17OA E/H<br />
*# P17OB E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] A E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] B E/H<br />
*# Omp-C1-reporter A X/P<br />
*# Omp-C1-reporter B X/P<br />
*# Omp-C1-reporter C X/P<br />
*# Omp-C1-reporter D X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] A X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] B X/P<br />
*# Death-ComA 5min ligation A X/P<br />
*# Death-ComA 5min ligation B X/P<br />
*# Death-ComA overnight ligation A X/P<br />
*# Death-ComA overnight ligation B X/P<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
Gel2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Death-ComP A X/P<br />
*# Death-ComP B X/P<br />
*# Cph8 A X/P<br />
*# Cph8 B X/P<br />
<br />
<br />
-All had desired insert except for lanes 8-11 (OmpR-C1-reporter) in Gel 1 and lanes 4, 5 (CpH8) in Gel 2.<br />
<br />
*Gel purified:<br />
*# [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
*# [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
*# Death-CompA E/X<br />
*# Death-ComP E/X<br />
*# [[Melbourne/BBa_J61035|P4 8J]] A E/S<br />
*# [[Melbourne/BBa_J61035|P4 8J]] B E/S<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 1hour ligation method at room temperature:<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-CompA E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-ComP E/X<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] above and plated.<br />
<br />
<br />
<font size=3><b>18 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies each from transformants of 17/9 (2 from Death-ComA).<br />
<br />
<br />
<font size=3><b>19 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] all cultures from 18/9.<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP 1 E/S<br />
*# Gen-RBS-ComP 2 E/S<br />
*# Gen-RBS-ComP 3 E/S<br />
*# Gen-RBS-ComP 4 E/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 1 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 2 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 3 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 4 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 1 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 2 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 3 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 4 X/S<br />
*# Gen-RBS-ComA 1 E/S<br />
*# Gen-RBS-ComA 2 E/S<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
-Ran at 100V for 45min.<br />
-Lanes 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, and 15 had desired bands. <br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP E/S 3.9kb<br />
*#<br />
*# Gen-RBS-ComA E/S 2.1kb <font color=green> Had no DNA </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 2.2kb <font color=green> Took wrong fragment </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S 2.2kb<br />
*#<br />
*# L3 X/P 550bp <font color=green> Ran off </font><br />
*#<br />
*# P17O S/P 2kb<br />
*#<br />
*# <br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] with 5min method: <br />
<br />
*# Gen-RBS-ComP E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/K<br />
<br />
<font size=3><b>20 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>21 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.<br />
<br />
<font size=3><b>22 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9). <br />
*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.<br />
<br />
==Week 14==<br />
<font size=3><b>23 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>24 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*No Growth observed from cultures BU ABCD of 22/9.<br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]]:<br />
*# Gen-RBS-ComP-ter <br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter <br />
*# [[Melbourne/BBa_J61035|P4 8J]] 1 (22/9)<br />
*# [[Melbourne/BBa_J61035|P4 8J]] 2 (22/9)<br />
*# PJS34 1 (22/9)<br />
*# PJS34 2 (22/9)<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] <br />
*# Gen-RBS-ComP X/P (19/9)<br />
*# Gen-RBS-ComP-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (19/9)<br />
*# PJS34 X/P<br />
*# Gen-RBS-ComA E/S<br />
*# L3 E/X<br />
<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for diagnosing:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP X/P<br />
*# Gen-RBS-ComP-ter X/P A<br />
*# Gen-RBS-ComP-ter X/P B<br />
*# Gen-RBS-ComP-ter X/P C<br />
*# Gen-RBS-ComP-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P B<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P C<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S A<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S B<br />
*# PJS34 X/P A<br />
*# PJS34 X/P B<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP-ter X/P A <font color=green> band at 700bp </font> (expected 4.6kb band)<br />
*#<br />
*# Gen-RBS-ComP-ter X/P D <font color=green> band at 700bp </font><br />
*#<br />
*# Gen-RBS-ComA 3 E/S <font color=green> 2.2kb band taken. </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)<br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9) <font color=green> took 3kb band </font><br />
*#<br />
*# L3 E/X took 2kb band<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10min method:<br />
*# TetR to RBS-ComP-Ter<br />
*# Gen-RBS-ComA to Ter<br />
*# OmpR-c1 to L3<br />
*# TetR to RBS-[[Melbourne/BBa_I15008|P2 21A]]<br />
<br />
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were create for:<br />
*# PJS34 1 + 2<br />
*# Gen-RBS-ComP-Ter A <font color=green> not sure </font><br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] A <font color=green> not sure </font><br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] ligations and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>25 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.<br />
<br />
<br />
<font size=3><b>26 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] the following<br />
**TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]]-ter A,B,C and D<br />
**TetR-RBS-ComP-ter A,B,C and D<br />
**Gen-RBS-ComA-ter A,B,C and D<br />
**OmpR-cI-L3 A,B,C and D<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] (all were X/P digests):<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 1<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 2<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 3<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] <br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] A<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] B<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] C<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] D<br />
*#<br />
*#<br />
*#<br />
*#<br />
*#<br />
*# Gen-RBS-ComA-ter D<br />
*# Gen-RBS-ComA-ter C<br />
*# Gen-RBS-ComA-ter B<br />
*# Gen-RBS-ComA-ter A<br />
*# Gen-RBS-ComA<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Omp-c1-L3 A<br />
*# Omp-c1-L3 B<br />
*# Omp-c1-L3 C<br />
*# Omp-c1-L3 D<br />
*# Death-ComP A<br />
*# RBS-ComP<br />
*# Gen-RBS-ComP-ter A<br />
*# Gen-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter A<br />
*# TetR-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter C<br />
*# TetR-RBS-ComP-ter D<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
<font size=3><b>27 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>28 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>29 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 15==<br />
<font size=3><b>30 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>1 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]<br />
<br />
Gel 1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComA-ter X/P 850bp<br />
*#<br />
*# ComA X/P 750bp <font color=green> purified </font><br />
*#<br />
*# Gen-RBS-ComA 2 X/P 760bp<br />
*#<br />
*# Gen-RBS- ComA 3 X/P 760bp <font color=green> purified although very low yield</font><br />
*#<br />
*#Gen-RBS- ComA 2 E/S 2.2kb<br />
*#<br />
*# Gen-RBS-ComA 3 E/S 2.2kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
Gel 2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# <br />
*# ComP X/P 2.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P 726bp<br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P 726bp <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] S/P 5.4kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] E/S 1kb <font color=green> purified </font><br />
*#<br />
*# L3 X/P (9/9) 550bp<br />
*#<br />
*# [[Melbourne/BBa_P1010_A|P3 20I]] X/P 2.1kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_J61035|P4 8J]] S/P 3.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*Also purified [[Melbourne/BBa_J61035|P4 8J]] E/S (1.5kb) on another gel.<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# Gen-RBS-ComA (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)<br />
*# ComA (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_P1010_A|P3 20I]] (X/P)<br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>2 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>3 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
*All plates had colonies from 2/10.<br />
*4 colonies were picked from each plate of 2/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<br />
<font size=3><b>4 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 3/10. Added 250ul of Nuclease free-water/TE buffer instead of 50ul in the final elution step. The DNA may be too dilute. <br />
<br />
<font size=3><b>5 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>6 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 16==<br />
<font size=3><b>7 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>8 Oct 2007 <br />
</b><br />
</font><BR><br />
*[[Melbourne/Diagnostic Digest| Digested]] 10uL of minipreps from 4/10 in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(X/P) (confirmed)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA2(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA3(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA4(X/P)<br />
# ComP (X/P) <br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (X/P)(confirmed)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP2 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP3 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP4 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP1 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP2 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP3 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP4 (X/P)<br />
# 1 kb+ ladder<br />
<br />
Gel 2:<br />
# 1 kb+ ladder<br />
#<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(E/S) -> gel purified 2.2kb fragment<br />
#<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment<br />
#<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# RA1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GAT<br />
*# RP1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GPT<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<font size=3><b>9 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*All plates had colonies from 8/10.<br />
*4 colonies were picked from each plate of 8/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<font size=3><b>10 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 9/10. <br />
*[[Melbourne/Diagnostic Digest| Digested]] minipreps in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# RA1(X/P) <br />
# GAT1 (X/P)<br />
# GAT2 (X/P)(confirmed)<br />
# GAT3 (X/P)<br />
# GAT4 (X/P)<br />
# ComP (X/P) <br />
# RP1 (X/P)<br />
# GPT1 (X/P)<br />
# GPT2 (X/P)<br />
# GPT3 (X/P)<br />
# GPT4 (X/P)(confirmed)<br />
# 1 kb+ ladder<br />
<br />
GAT2= GenRBS-ComA-ter<br />
GPT4= GenRBS-ComP-ter -> need sequence confirmation<br />
<br />
<br />
<font size=3><b>11 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>12 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>13 Oct 2007 <br />
</b><br />
</font><BR></div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_13-16
Melbourne/Lab Notebook Weeks 13-16
2007-10-25T05:06:27Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne| <team home page>]]<br />
<br />
==Week 13==<br />
<font size=3><b>16 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>17 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] 2 cultures from each of the transformations on 12/9 and 4 cultures from Omp-C1-reporter.<br />
*[[Melbourne/Diagnostic Digest| Digested]] these and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# P17OA E/H<br />
*# P17OB E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] A E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] B E/H<br />
*# Omp-C1-reporter A X/P<br />
*# Omp-C1-reporter B X/P<br />
*# Omp-C1-reporter C X/P<br />
*# Omp-C1-reporter D X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] A X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] B X/P<br />
*# Death-ComA 5min ligation A X/P<br />
*# Death-ComA 5min ligation B X/P<br />
*# Death-ComA overnight ligation A X/P<br />
*# Death-ComA overnight ligation B X/P<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
Gel2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Death-ComP A X/P<br />
*# Death-ComP B X/P<br />
*# Cph8 A X/P<br />
*# Cph8 B X/P<br />
<br />
<br />
-All had desired insert except for lanes 8-11 (OmpR-C1-reporter) in Gel 1 and lanes 4, 5 (CpH8) in Gel 2.<br />
<br />
*Gel purified:<br />
*# [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
*# [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
*# Death-CompA E/X<br />
*# Death-ComP E/X<br />
*# [[Melbourne/BBa_J61035|P4 8J]] A E/S<br />
*# [[Melbourne/BBa_J61035|P4 8J]] B E/S<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 1hour ligation method at room temperature:<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-CompA E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-ComP E/X<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] above and plated.<br />
<br />
<br />
<font size=3><b>18 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies each from transformants of 17/9 (2 from Death-ComA).<br />
<br />
<br />
<font size=3><b>19 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] all cultures from 18/9.<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP 1 E/S<br />
*# Gen-RBS-ComP 2 E/S<br />
*# Gen-RBS-ComP 3 E/S<br />
*# Gen-RBS-ComP 4 E/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 1 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 2 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 3 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 4 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 1 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 2 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 3 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 4 X/S<br />
*# Gen-RBS-ComA 1 E/S<br />
*# Gen-RBS-ComA 2 E/S<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
-Ran at 100V for 45min.<br />
-Lanes 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, and 15 had desired bands. <br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP E/S 3.9kb<br />
*#<br />
*# Gen-RBS-ComA E/S 2.1kb <font color=green> Had no DNA </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 2.2kb <font color=green> Took wrong fragment </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S 2.2kb<br />
*#<br />
*# L3 X/P 550bp <font color=green> Ran off </font><br />
*#<br />
*# P17O S/P 2kb<br />
*#<br />
*# <br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] with 5min method: <br />
<br />
*# Gen-RBS-ComP E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/K<br />
<br />
<font size=3><b>20 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>21 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.<br />
<br />
<font size=3><b>22 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9). <br />
*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.<br />
<br />
==Week 14==<br />
<font size=3><b>23 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>24 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*No Growth observed from cultures BU ABCD of 22/9.<br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]]:<br />
*# Gen-RBS-ComP-ter <br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter <br />
*# [[Melbourne/BBa_J61035|P4 8J]] 1 (22/9)<br />
*# [[Melbourne/BBa_J61035|P4 8J]] 2 (22/9)<br />
*# PJS34 1 (22/9)<br />
*# PJS34 2 (22/9)<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] <br />
*# Gen-RBS-ComP X/P (19/9)<br />
*# Gen-RBS-ComP-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (19/9)<br />
*# PJS34 X/P<br />
*# Gen-RBS-ComA E/S<br />
*# L3 E/X<br />
<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for diagnosing:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP X/P<br />
*# Gen-RBS-ComP-ter X/P A<br />
*# Gen-RBS-ComP-ter X/P B<br />
*# Gen-RBS-ComP-ter X/P C<br />
*# Gen-RBS-ComP-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P B<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P C<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S A<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S B<br />
*# PJS34 X/P A<br />
*# PJS34 X/P B<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP-ter X/P A <font color=green> band at 700bp </font> (expected 4.6kb band)<br />
*#<br />
*# Gen-RBS-ComP-ter X/P D <font color=green> band at 700bp </font><br />
*#<br />
*# Gen-RBS-ComA 3 E/S <font color=green> 2.2kb band taken. </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)<br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9) <font color=green> took 3kb band </font><br />
*#<br />
*# L3 E/X took 2kb band<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10min method:<br />
*# TetR to RBS-ComP-Ter<br />
*# Gen-RBS-ComA to Ter<br />
*# OmpR-c1 to L3<br />
*# TetR to RBS-[[Melbourne/BBa_I15008|P2 21A]]<br />
<br />
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were create for:<br />
*# PJS34 1 + 2<br />
*# Gen-RBS-ComP-Ter A <font color=green> not sure </font><br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] A <font color=green> not sure </font><br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] ligations and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>25 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.<br />
<br />
<br />
<font size=3><b>26 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] the following<br />
**TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]]-ter A,B,C and D<br />
**TetR-RBS-ComP-ter A,B,C and D<br />
**Gen-RBS-ComA-ter A,B,C and D<br />
**OmpR-cI-L3 A,B,C and D<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] (all were X/P digests):<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 1<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 2<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 3<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] <br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] A<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] B<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] C<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] D<br />
*#<br />
*#<br />
*#<br />
*#<br />
*#<br />
*# Gen-RBS-ComA-ter D<br />
*# Gen-RBS-ComA-ter C<br />
*# Gen-RBS-ComA-ter B<br />
*# Gen-RBS-ComA-ter A<br />
*# Gen-RBS-ComA<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Omp-c1-L3 A<br />
*# Omp-c1-L3 B<br />
*# Omp-c1-L3 C<br />
*# Omp-c1-L3 D<br />
*# Death-ComP A<br />
*# RBS-ComP<br />
*# Gen-RBS-ComP-ter A<br />
*# Gen-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter A<br />
*# TetR-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter C<br />
*# TetR-RBS-ComP-ter D<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
<font size=3><b>27 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>28 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>29 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 15==<br />
<font size=3><b>30 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>1 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]<br />
<br />
Gel 1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComA-ter X/P 850bp<br />
*#<br />
*# ComA X/P 750bp <font color=green> purified </font><br />
*#<br />
*# Gen-RBS-ComA 2 X/P 760bp<br />
*#<br />
*# Gen-RBS- ComA 3 X/P 760bp <font color=green> purified although very low yield</font><br />
*#<br />
*#Gen-RBS- ComA 2 E/S 2.2kb<br />
*#<br />
*# Gen-RBS-ComA 3 E/S 2.2kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
Gel 2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# <br />
*# ComP X/P 2.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P 726bp<br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P 726bp <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] S/P 5.4kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] E/S 1kb <font color=green> purified </font><br />
*#<br />
*# L3 X/P (9/9) 550bp<br />
*#<br />
*# [[Melbourne/BBa_P1010_A|P3 20I]] X/P 2.1kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_J61035|P4 8J]] S/P 3.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*Also purified [[Melbourne/BBa_J61035|P4 8J]] E/S (1.5kb) on another gel.<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# Gen-RBS-ComA (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)<br />
*# ComA (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_P1010_A|P3 20I]] (X/P)<br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>2 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>3 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
*All plates had colonies from 2/10.<br />
*4 colonies were picked from each plate of 2/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<br />
<font size=3><b>4 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 3/10. Added 250ul of Nuclease free-water/TE buffer instead of 50ul in the final elution step. The DNA may be too dilute. <br />
<br />
<font size=3><b>5 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>6 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 16==<br />
<font size=3><b>7 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>8 Oct 2007 <br />
</b><br />
</font><BR><br />
*[[Melbourne/Diagnostic Digest| Digested]] 10uL of minipreps from 4/10 in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(X/P) (confirmed)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA2(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA3(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA4(X/P)<br />
# ComP (X/P) <br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (X/P)(confirmed)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP2 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP3 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP4 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP1 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP2 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP3 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP4 (X/P)<br />
# 1 kb+ ladder<br />
<br />
Gel 2:<br />
# 1 kb+ ladder<br />
#<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(E/S) -> gel purified 2.2kb fragment<br />
#<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment<br />
#<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# RA1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GAT<br />
*# RP1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GPT<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<font size=3><b>9 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*All plates had colonies from 8/10.<br />
*4 colonies were picked from each plate of 8/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<font size=3><b>10 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 9/10. <br />
*[[Melbourne/Diagnostic Digest| Digested]] minipreps in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# RA1(X/P) <br />
# GAT1 (X/P)<br />
# GAT2 (X/P)(confirmed)<br />
# GAT3 (X/P)<br />
# GAT4 (X/P)<br />
# ComP (X/P) <br />
# RP1 (X/P)<br />
# GPT1 (X/P)<br />
# GPT2 (X/P)<br />
# GPT3 (X/P)<br />
# GPT4 (X/P)(confirmed)<br />
# 1 kb+ ladder<br />
<br />
<br />
<font size=3><b>11 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>12 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>13 Oct 2007 <br />
</b><br />
</font><BR></div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_13-16
Melbourne/Lab Notebook Weeks 13-16
2007-10-25T05:01:00Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne| <team home page>]]<br />
<br />
==Week 13==<br />
<font size=3><b>16 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>17 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] 2 cultures from each of the transformations on 12/9 and 4 cultures from Omp-C1-reporter.<br />
*[[Melbourne/Diagnostic Digest| Digested]] these and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# P17OA E/H<br />
*# P17OB E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] A E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] B E/H<br />
*# Omp-C1-reporter A X/P<br />
*# Omp-C1-reporter B X/P<br />
*# Omp-C1-reporter C X/P<br />
*# Omp-C1-reporter D X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] A X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] B X/P<br />
*# Death-ComA 5min ligation A X/P<br />
*# Death-ComA 5min ligation B X/P<br />
*# Death-ComA overnight ligation A X/P<br />
*# Death-ComA overnight ligation B X/P<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
Gel2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Death-ComP A X/P<br />
*# Death-ComP B X/P<br />
*# Cph8 A X/P<br />
*# Cph8 B X/P<br />
<br />
<br />
-All had desired insert except for lanes 8-11 (OmpR-C1-reporter) in Gel 1 and lanes 4, 5 (CpH8) in Gel 2.<br />
<br />
*Gel purified:<br />
*# [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
*# [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
*# Death-CompA E/X<br />
*# Death-ComP E/X<br />
*# [[Melbourne/BBa_J61035|P4 8J]] A E/S<br />
*# [[Melbourne/BBa_J61035|P4 8J]] B E/S<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 1hour ligation method at room temperature:<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-CompA E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-ComP E/X<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] above and plated.<br />
<br />
<br />
<font size=3><b>18 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies each from transformants of 17/9 (2 from Death-ComA).<br />
<br />
<br />
<font size=3><b>19 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] all cultures from 18/9.<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP 1 E/S<br />
*# Gen-RBS-ComP 2 E/S<br />
*# Gen-RBS-ComP 3 E/S<br />
*# Gen-RBS-ComP 4 E/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 1 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 2 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 3 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 4 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 1 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 2 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 3 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 4 X/S<br />
*# Gen-RBS-ComA 1 E/S<br />
*# Gen-RBS-ComA 2 E/S<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
-Ran at 100V for 45min.<br />
-Lanes 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, and 15 had desired bands. <br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP E/S 3.9kb<br />
*#<br />
*# Gen-RBS-ComA E/S 2.1kb <font color=green> Had no DNA </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 2.2kb <font color=green> Took wrong fragment </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S 2.2kb<br />
*#<br />
*# L3 X/P 550bp <font color=green> Ran off </font><br />
*#<br />
*# P17O S/P 2kb<br />
*#<br />
*# <br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] with 5min method: <br />
<br />
*# Gen-RBS-ComP E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/K<br />
<br />
<font size=3><b>20 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>21 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.<br />
<br />
<font size=3><b>22 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9). <br />
*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.<br />
<br />
==Week 14==<br />
<font size=3><b>23 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>24 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*No Growth observed from cultures BU ABCD of 22/9.<br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]]:<br />
*# Gen-RBS-ComP-ter <br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter <br />
*# [[Melbourne/BBa_J61035|P4 8J]] 1 (22/9)<br />
*# [[Melbourne/BBa_J61035|P4 8J]] 2 (22/9)<br />
*# PJS34 1 (22/9)<br />
*# PJS34 2 (22/9)<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] <br />
*# Gen-RBS-ComP X/P (19/9)<br />
*# Gen-RBS-ComP-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (19/9)<br />
*# PJS34 X/P<br />
*# Gen-RBS-ComA E/S<br />
*# L3 E/X<br />
<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for diagnosing:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP X/P<br />
*# Gen-RBS-ComP-ter X/P A<br />
*# Gen-RBS-ComP-ter X/P B<br />
*# Gen-RBS-ComP-ter X/P C<br />
*# Gen-RBS-ComP-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P B<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P C<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S A<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S B<br />
*# PJS34 X/P A<br />
*# PJS34 X/P B<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP-ter X/P A <font color=green> band at 700bp </font> (expected 4.6kb band)<br />
*#<br />
*# Gen-RBS-ComP-ter X/P D <font color=green> band at 700bp </font><br />
*#<br />
*# Gen-RBS-ComA 3 E/S <font color=green> 2.2kb band taken. </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)<br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9) <font color=green> took 3kb band </font><br />
*#<br />
*# L3 E/X took 2kb band<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10min method:<br />
*# TetR to RBS-ComP-Ter<br />
*# Gen-RBS-ComA to Ter<br />
*# OmpR-c1 to L3<br />
*# TetR to RBS-[[Melbourne/BBa_I15008|P2 21A]]<br />
<br />
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were create for:<br />
*# PJS34 1 + 2<br />
*# Gen-RBS-ComP-Ter A <font color=green> not sure </font><br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] A <font color=green> not sure </font><br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] ligations and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>25 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.<br />
<br />
<br />
<font size=3><b>26 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] the following<br />
**TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]]-ter A,B,C and D<br />
**TetR-RBS-ComP-ter A,B,C and D<br />
**Gen-RBS-ComA-ter A,B,C and D<br />
**OmpR-cI-L3 A,B,C and D<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] (all were X/P digests):<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 1<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 2<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 3<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] <br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] A<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] B<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] C<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] D<br />
*#<br />
*#<br />
*#<br />
*#<br />
*#<br />
*# Gen-RBS-ComA-ter D<br />
*# Gen-RBS-ComA-ter C<br />
*# Gen-RBS-ComA-ter B<br />
*# Gen-RBS-ComA-ter A<br />
*# Gen-RBS-ComA<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Omp-c1-L3 A<br />
*# Omp-c1-L3 B<br />
*# Omp-c1-L3 C<br />
*# Omp-c1-L3 D<br />
*# Death-ComP A<br />
*# RBS-ComP<br />
*# Gen-RBS-ComP-ter A<br />
*# Gen-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter A<br />
*# TetR-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter C<br />
*# TetR-RBS-ComP-ter D<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
<font size=3><b>27 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>28 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>29 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 15==<br />
<font size=3><b>30 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>1 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]<br />
<br />
Gel 1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComA-ter X/P 850bp<br />
*#<br />
*# ComA X/P 750bp <font color=green> purified </font><br />
*#<br />
*# Gen-RBS-ComA 2 X/P 760bp<br />
*#<br />
*# Gen-RBS- ComA 3 X/P 760bp <font color=green> purified although very low yield</font><br />
*#<br />
*#Gen-RBS- ComA 2 E/S 2.2kb<br />
*#<br />
*# Gen-RBS-ComA 3 E/S 2.2kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
Gel 2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# <br />
*# ComP X/P 2.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P 726bp<br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P 726bp <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] S/P 5.4kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] E/S 1kb <font color=green> purified </font><br />
*#<br />
*# L3 X/P (9/9) 550bp<br />
*#<br />
*# [[Melbourne/BBa_P1010_A|P3 20I]] X/P 2.1kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_J61035|P4 8J]] S/P 3.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*Also purified [[Melbourne/BBa_J61035|P4 8J]] E/S (1.5kb) on another gel.<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# Gen-RBS-ComA (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)<br />
*# ComA (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_P1010_A|P3 20I]] (X/P)<br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>2 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>3 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
*All plates had colonies from 2/10.<br />
*4 colonies were picked from each plate of 2/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<br />
<font size=3><b>4 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 3/10. Added 250ul of Nuclease free-water/TE buffer instead of 50ul in the final elution step. The DNA may be too dilute. <br />
<br />
<font size=3><b>5 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>6 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 16==<br />
<font size=3><b>7 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>8 Oct 2007 <br />
</b><br />
</font><BR><br />
*[[Melbourne/Diagnostic Digest| Digested]] 10uL of minipreps from 4/10 in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(X/P) (confirmed)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA2(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA3(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA4(X/P)<br />
# ComP (X/P) <br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (X/P)(confirmed)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP2 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP3 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP4 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP1 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP2 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP3 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP4 (X/P)<br />
# 1 kb+ ladder<br />
<br />
Gel 2:<br />
# 1 kb+ ladder<br />
#<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(E/S) -> gel purified 2.2kb fragment<br />
#<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment<br />
#<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# RA1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GAT<br />
*# RP1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GPT<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<font size=3><b>9 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>10 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>11 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>12 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>13 Oct 2007 <br />
</b><br />
</font><BR></div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_13-16
Melbourne/Lab Notebook Weeks 13-16
2007-10-25T04:54:03Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne| <team home page>]]<br />
<br />
==Week 13==<br />
<font size=3><b>16 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>17 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] 2 cultures from each of the transformations on 12/9 and 4 cultures from Omp-C1-reporter.<br />
*[[Melbourne/Diagnostic Digest| Digested]] these and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# P17OA E/H<br />
*# P17OB E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] A E/H<br />
*# [[Melbourne/BBa_C0051|P1 5G]] B E/H<br />
*# Omp-C1-reporter A X/P<br />
*# Omp-C1-reporter B X/P<br />
*# Omp-C1-reporter C X/P<br />
*# Omp-C1-reporter D X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] A X/P<br />
*# [[Melbourne/BBa_I15009|P2 21C]] B X/P<br />
*# Death-ComA 5min ligation A X/P<br />
*# Death-ComA 5min ligation B X/P<br />
*# Death-ComA overnight ligation A X/P<br />
*# Death-ComA overnight ligation B X/P<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
Gel2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Death-ComP A X/P<br />
*# Death-ComP B X/P<br />
*# Cph8 A X/P<br />
*# Cph8 B X/P<br />
<br />
<br />
-All had desired insert except for lanes 8-11 (OmpR-C1-reporter) in Gel 1 and lanes 4, 5 (CpH8) in Gel 2.<br />
<br />
*Gel purified:<br />
*# [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
*# [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
*# Death-CompA E/X<br />
*# Death-ComP E/X<br />
*# [[Melbourne/BBa_J61035|P4 8J]] A E/S<br />
*# [[Melbourne/BBa_J61035|P4 8J]] B E/S<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 1hour ligation method at room temperature:<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15008|P2 21A]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + [[Melbourne/BBa_I15009|P2 21C]] E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-CompA E/X<br />
**[[Melbourne/BBa_J61035|P4 8J]] E/S + Death-ComP E/X<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] above and plated.<br />
<br />
<br />
<font size=3><b>18 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies each from transformants of 17/9 (2 from Death-ComA).<br />
<br />
<br />
<font size=3><b>19 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] all cultures from 18/9.<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP 1 E/S<br />
*# Gen-RBS-ComP 2 E/S<br />
*# Gen-RBS-ComP 3 E/S<br />
*# Gen-RBS-ComP 4 E/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 1 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 2 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 3 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] 4 X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 1 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 2 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 3 X/S<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] 4 X/S<br />
*# Gen-RBS-ComA 1 E/S<br />
*# Gen-RBS-ComA 2 E/S<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
-Ran at 100V for 45min.<br />
-Lanes 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, and 15 had desired bands. <br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP E/S 3.9kb<br />
*#<br />
*# Gen-RBS-ComA E/S 2.1kb <font color=green> Had no DNA </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 2.2kb <font color=green> Took wrong fragment </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S 2.2kb<br />
*#<br />
*# L3 X/P 550bp <font color=green> Ran off </font><br />
*#<br />
*# P17O S/P 2kb<br />
*#<br />
*# <br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*# [[Melbourne/BBa_C0051|P1 5G]] E/X 3.3kb<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] with 5min method: <br />
<br />
*# Gen-RBS-ComP E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] E/S into [[Melbourne/BBa_C0051|P1 5G]] E/X -survives on G/K<br />
<br />
<font size=3><b>20 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>21 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.<br />
<br />
<font size=3><b>22 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9). <br />
*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.<br />
<br />
==Week 14==<br />
<font size=3><b>23 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>24 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*No Growth observed from cultures BU ABCD of 22/9.<br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]]:<br />
*# Gen-RBS-ComP-ter <br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter <br />
*# [[Melbourne/BBa_J61035|P4 8J]] 1 (22/9)<br />
*# [[Melbourne/BBa_J61035|P4 8J]] 2 (22/9)<br />
*# PJS34 1 (22/9)<br />
*# PJS34 2 (22/9)<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] <br />
*# Gen-RBS-ComP X/P (19/9)<br />
*# Gen-RBS-ComP-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (19/9)<br />
*# PJS34 X/P<br />
*# Gen-RBS-ComA E/S<br />
*# L3 E/X<br />
<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for diagnosing:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComP X/P<br />
*# Gen-RBS-ComP-ter X/P A<br />
*# Gen-RBS-ComP-ter X/P B<br />
*# Gen-RBS-ComP-ter X/P C<br />
*# Gen-RBS-ComP-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P A<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P B<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P C<br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P D<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S A<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S B<br />
*# PJS34 X/P A<br />
*# PJS34 X/P B<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
[[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*#<br />
*# Gen-RBS-ComP-ter X/P A <font color=green> band at 700bp </font> (expected 4.6kb band)<br />
*#<br />
*# Gen-RBS-ComP-ter X/P D <font color=green> band at 700bp </font><br />
*#<br />
*# Gen-RBS-ComA 3 E/S <font color=green> 2.2kb band taken. </font><br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)<br />
*#<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9) <font color=green> took 3kb band </font><br />
*#<br />
*# L3 E/X took 2kb band<br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10min method:<br />
*# TetR to RBS-ComP-Ter<br />
*# Gen-RBS-ComA to Ter<br />
*# OmpR-c1 to L3<br />
*# TetR to RBS-[[Melbourne/BBa_I15008|P2 21A]]<br />
<br />
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were create for:<br />
*# PJS34 1 + 2<br />
*# Gen-RBS-ComP-Ter A <font color=green> not sure </font><br />
*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] A <font color=green> not sure </font><br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] ligations and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>25 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.<br />
<br />
<br />
<font size=3><b>26 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] the following<br />
**TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]]-ter A,B,C and D<br />
**TetR-RBS-ComP-ter A,B,C and D<br />
**Gen-RBS-ComA-ter A,B,C and D<br />
**OmpR-cI-L3 A,B,C and D<br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] (all were X/P digests):<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 1<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 2<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 3<br />
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] <br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] A<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] B<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] C<br />
*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] D<br />
*#<br />
*#<br />
*#<br />
*#<br />
*#<br />
*# Gen-RBS-ComA-ter D<br />
*# Gen-RBS-ComA-ter C<br />
*# Gen-RBS-ComA-ter B<br />
*# Gen-RBS-ComA-ter A<br />
*# Gen-RBS-ComA<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Omp-c1-L3 A<br />
*# Omp-c1-L3 B<br />
*# Omp-c1-L3 C<br />
*# Omp-c1-L3 D<br />
*# Death-ComP A<br />
*# RBS-ComP<br />
*# Gen-RBS-ComP-ter A<br />
*# Gen-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter A<br />
*# TetR-RBS-ComP-ter B<br />
*# TetR-RBS-ComP-ter C<br />
*# TetR-RBS-ComP-ter D<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
<font size=3><b>27 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>28 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>29 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 15==<br />
<font size=3><b>30 Sept 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>1 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]<br />
<br />
Gel 1:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# Gen-RBS-ComA-ter X/P 850bp<br />
*#<br />
*# ComA X/P 750bp <font color=green> purified </font><br />
*#<br />
*# Gen-RBS-ComA 2 X/P 760bp<br />
*#<br />
*# Gen-RBS- ComA 3 X/P 760bp <font color=green> purified although very low yield</font><br />
*#<br />
*#Gen-RBS- ComA 2 E/S 2.2kb<br />
*#<br />
*# Gen-RBS-ComA 3 E/S 2.2kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
Gel 2:<br />
<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
*# <br />
*# ComP X/P 2.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] A X/P 726bp<br />
*#<br />
*# [[Melbourne/BBa_I15008|P2 21A]] B X/P 726bp <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] S/P 5.4kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_Q04510|P2 13K]] E/S 1kb <font color=green> purified </font><br />
*#<br />
*# L3 X/P (9/9) 550bp<br />
*#<br />
*# [[Melbourne/BBa_P1010_A|P3 20I]] X/P 2.1kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/BBa_J61035|P4 8J]] S/P 3.5kb <font color=green> purified </font><br />
*#<br />
*# [[Melbourne/primary DNA marker|DNA ladder]]<br />
<br />
<br />
*Also purified [[Melbourne/BBa_J61035|P4 8J]] E/S (1.5kb) on another gel.<br />
<br />
<br />
*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.<br />
*# Gen-RBS-ComA (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)<br />
*# ComA (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)<br />
*# ComP (X/P) to [[Melbourne/BBa_P1010_A|P3 20I]] (X/P)<br />
<br />
<br />
*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.<br />
<br />
<br />
<br />
<font size=3><b>2 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>3 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
*All plates had colonies from 2/10.<br />
*4 colonies were picked from each plate of 2/10 and [[Melbourne/Growing up cells|Liquid cultured]].<br />
<br />
<br />
<font size=3><b>4 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 3/10. Added 250ul of Nuclease free-water/TE buffer instead of 50ul in the final elution step. The DNA may be too dilute. <br />
<br />
<font size=3><b>5 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>6 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
==Week 16==<br />
<font size=3><b>7 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>8 Oct 2007 <br />
</b><br />
</font><BR><br />
*[[Melbourne/Diagnostic Digest| Digested]] 10uL of minipreps from 4/10 in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:<br />
<br />
Gel1:<br />
# 1 kb+ ladder<br />
# ComA (X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(X/P) (confirmed)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA2(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA3(X/P)<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA4(X/P)<br />
# ComP (X/P) <br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (X/P)(confirmed)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP2 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP3 (X/P)<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP4 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP1 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP2 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP3 (X/P)<br />
# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP4 (X/P)<br />
# 1 kb+ ladder<br />
<br />
Gel 2:<br />
# 1 kb+ ladder<br />
#<br />
# ComA to [[Melbourne/BBa_J61035|P4 8J]] = RA1(E/S) -> gel purified 2.2kb fragment<br />
#<br />
# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment<br />
#<br />
<br />
<br />
<br />
<br />
<font size=3><b>9 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>10 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>11 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>12 Oct 2007 <br />
</b><br />
</font><BR><br />
<br />
<br />
<font size=3><b>13 Oct 2007 <br />
</b><br />
</font><BR></div>
Patricia
http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2007-10-25T04:39:37Z
<p>Patricia: </p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
=====Contact=====<br />
pat@illing.com.au<br />
<br />
=====Education=====<br />
I am currently completing my third year of a Bachelor of Science Degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me, iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse all our undertakings in the lab.<br />
<br />
=====Interests outside of science include:=====<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2007-10-25T04:38:44Z
<p>Patricia: </p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
=====Contact=====<br />
pat@illing.com.au<br />
<br />
=====Education=====<br />
I am currently completing my third year of a Bachelor of Science Degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse what we are doing and why.<br />
<br />
=====Interests outside of science include:=====<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2007-10-25T04:37:38Z
<p>Patricia: </p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
=Education=<br />
I am currently completing my third year of a Bachelor of Science Degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse what we are doing and why.<br />
<br />
=Interests outside of science include:=<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/User:Patricia
User:Patricia
2007-10-25T04:35:08Z
<p>Patricia: </p>
<hr />
<div>[[Melb:team|<Back to Melbourne Team Page>]] [[Melbourne|<Back to Melbourne Home Page>]]<br />
<br />
I am currently completing my third year of a Bachelor of Science Degree, majoring in Biochemistry and Molecular biology, and Cell Biology. Next year I intend to undertake an Honours year, most likely in <br />
Biochemistry, but I also have a keen interest in developmental biology.<br />
<br />
For me iGEM has been a chance to increase my competance and independence in the lab. The self-directed nature of our project has ensured that we have all gained the ability to closely analyse what we are doing and why.<br />
<br />
Interests outside of science include:<br />
*chocolate making<br />
*baking<br />
*SCUBA diving<br />
*swing dancing<br />
*music</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_created
Melbourne/Parts created
2007-10-21T10:59:54Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: <br />
{| border="1"<br />
|+ Parts Created by Melbourne [[Melbourne/parts created graphical| <GRAPHICAL>]]<br />
! Part Name !! Sub Parts !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status !! Sequenced<br />
|-<br />
! OmpR-EYFP<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_E0430 EYFP]|| 108+878 || pSB1A2 || 2079 || 3065 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 12+353+95 || pSB1A2 || 2079 || 2549 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 108+12+353+95 || pSB1A2 || 2079 || 2657 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-c1 inverter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_Q04510 c1 inverter] || 108+987 || pSB1A2 || 2079 || 3068 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! ComPalt<br />
| [[Melbourne/Lab BL Notebook/ComP'|ComP']]||2406 ||pSB1A3||2157||4563||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! ComAalt<br />
| [[Melbourne/Lab BL Notebook/ComA'|ComA']]||741 ||pSB1A3 ||2157 ||2898||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! GenRBS+ComPalt+ter<br />
| Gen[http://partsregistry.org/Part:BBa_B0034 RBS],[[Melbourne/Lab BL Notebook/ComP'|ComP']], [http://partsregistry.org/Part:BBa_B0014 double terminator]||2468 +1500Gen || pSB1AK3 ||3189||7.1kb||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
! GenRBS+ComAalt+ter<br />
| Gen[http://partsregistry.org/Part:BBa_B0034 RBS], [[Melbourne/Lab BL Notebook/ComA'|ComA']], [http://partsregistry.org/Part:BBa_B0014 double terminator]||803 +1500Gen ||pSB1AK3 ||3189|| 5.5kb ||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_created
Melbourne/Parts created
2007-10-21T10:59:18Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: <br />
{| border="1"<br />
|+ Parts Created by Melbourne [[Melbourne/parts created graphical| <GRAPHICAL>]]<br />
! Part Name !! Sub Parts !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status !! Sequenced<br />
|-<br />
! OmpR-EYFP<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_E0430 EYFP]|| 108+878 || pSB1A2 || 2079 || 3065 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 12+353+95 || pSB1A2 || 2079 || 2549 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 108+12+353+95 || pSB1A2 || 2079 || 2657 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-c1 inverter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_Q04510 c1 inverter] || 108+987 || pSB1A2 || 2079 || 3068 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! ComPalt<br />
| [[Melbourne/Lab BL Notebook/ComP'|ComP']]||2406 ||pSB1A3||2157||4563||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! ComAalt<br />
| [[Melbourne/Lab BL Notebook/ComA'|ComA']]||741 ||pSB1A3 ||2157 ||2898||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! GenRBS+ComPalt+ter<br />
| Gen[http://partsregistry.org/Part:BBa_B0034 RBS],[[Melbourne/Lab BL Notebook/ComP'|ComP']], double ter||2468 +1500Gen || pSB1AK3 ||3189||7.1kb||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
! GenRBS+ComAalt+ter<br />
| Gen[http://partsregistry.org/Part:BBa_B0034 RBS], [[Melbourne/Lab BL Notebook/ComA'|ComA']], double ter||803 +1500Gen ||pSB1AK3 ||3189|| 5.5kb ||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_created
Melbourne/Parts created
2007-10-21T10:58:03Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: <br />
{| border="1"<br />
|+ Parts Created by Melbourne [[Melbourne/parts created graphical| <GRAPHICAL>]]<br />
! Part Name !! Sub Parts !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status !! Sequenced<br />
|-<br />
! OmpR-EYFP<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_E0430 EYFP]|| 108+878 || pSB1A2 || 2079 || 3065 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 12+353+95 || pSB1A2 || 2079 || 2549 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 108+12+353+95 || pSB1A2 || 2079 || 2657 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-c1 inverter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_Q04510 c1 inverter] || 108+987 || pSB1A2 || 2079 || 3068 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! ComPalt<br />
| [[Melbourne/Lab BL Notebook/ComP'|ComP']]||2406 ||pSB1A3||2157||4563||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! ComAalt<br />
| [[Melbourne/Lab BL Notebook/ComA'|ComA']]||741 ||pSB1A3 ||2157 ||2898||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! GenRBS+ComPalt+ter<br />
| GenRBS,[[Melbourne/Lab BL Notebook/ComP'|ComP']], double ter||2468 +1500Gen || pSB1AK3 ||3189||7.1kb||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
! GenRBS+ComAalt+ter<br />
| GenRBS, [[Melbourne/Lab BL Notebook/ComA'|ComA']], double ter||803 +1500Gen ||pSB1AK3 ||3189|| 5.5kb ||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_created
Melbourne/Parts created
2007-10-21T10:56:56Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: <br />
{| border="1"<br />
|+ Parts Created by Melbourne [[Melbourne/parts created graphical| <GRAPHICAL>]]<br />
! Part Name !! Sub Parts !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status !! Sequenced<br />
|-<br />
! OmpR-EYFP<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_E0430 EYFP]|| 108+878 || pSB1A2 || 2079 || 3065 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 12+353+95 || pSB1A2 || 2079 || 2549 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-LacZ reporter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_B0034 RBS] - [http://partsregistry.org/Part:BBa_E0033 LacZ alpha] - [http://partsregistry.org/Part:BBa_B0014 double terminator] || 108+12+353+95 || pSB1A2 || 2079 || 2657 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! OmpR-c1 inverter<br />
| [http://partsregistry.org/Part:BBa_R0082 OmpR] - [http://partsregistry.org/Part:BBa_Q04510 c1 inverter] || 108+987 || pSB1A2 || 2079 || 3068 || Amp || Glycerol stocks in -80 || yes<br />
|-<br />
! ComPalt<br />
| [[Melbourne/Lab BL Notebook/ComP'|ComP']]||2406 ||pSB1A3||2157||4563||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! ComAalt<br />
| [[Melbourne/Lab BL Notebook/ComA'|ComA']]||741 ||pSB1A3 ||2157 ||2898||Amp|| Glycerol stocks in -80 || no<br />
|-<br />
! GenRBS+ComPalt+ter<br />
| [[Melbourne/Lab BL Notebook/ComP'|ComP']]||2468 +1500Gen || pSB1AK3 ||3189||7.1kb||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
! GenRBS+ComAalt+ter<br />
| [[Melbourne/Lab BL Notebook/ComA'|ComA']]||803 +1500Gen ||pSB1AK3 ||3189|| 5.5kb ||Amp, Kan, Gen|| Miniprepped || no<br />
|-<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook
Melbourne/Lab Notebook
2007-09-27T00:31:59Z
<p>Patricia: /* 26 Sept 2007 */</p>
<hr />
<div>[[Melbourne|<Back to team home page>]] <br />
<br />
==Week 1==<br />
*25 June 2007: Prepared LB agar plates Amp & Kana.<br />
*25 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melbourne/Transformation Protocol shorter|Transformed (shorter protocol)]] into competent DH5alpha cells.<br />
*#[[Melbourne/BBa_I15008|'''P2 21A''']] - (BBa_I15008, ho1, Kan) -> No colonies on plates<br />
*#[[Melbourne/BBa_I15009|'''P2 21C''']] - (BBa_I15009, PcyA, Kan) ->No colonies on plates<br />
*#[[Melbourne/BBa_R0084|'''P1 11H''']] - (BBa_R0084, OmpR positive promoter, Amp)->Small number of colonies.<br />
<br />
<br />
<br />
*26 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034, RBS, Amp)<br />
*#[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051, c1 protein, Amp)<br />
*#[[Melbourne/BBa_B0010|'''P2 3P''']] (BBa_B0010, Terminator, Amp)<br />
<br />
*26 June 2007: Streaked the following cells:<br />
*#[[Melbourne/pJS010|'''pJS010''']] (from solid agar, Amp)<br />
*#[[Melbourne/Fusion|'''Fusion protein''']] (from glycerol stock, Amp?)<br />
*#[[Melbourne/BBa_I15010|'''BBa_I15010''']] (from solid agar, Kan)<br />
<br />
*27 June 2007: [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#[[Melbourne/BBa_I15008|'''P2 21A''']] (Kan)<br />
*#[[Melbourne/BBa_I15009|'''P2 21C''']] (Kan)<br />
<br />
<br />
====Liquid culture====<br />
#[[Melb:Growing up cells|Prepared]] 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).<br />
#Aliquoted 5mL Amp LB into 6 50mL falcon tubes<br />
#To the Amp LB aliquots single transformed colonies from plates of the following were introduced:<br />
#*[[Melbourne/pJS010|'''pJS010''']]<br />
#*[[Melbourne/Fusion|'''Fusion''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']]<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']]<br />
#*[[Melbourne/BBa_B0010|'''P2 3P''']]<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']]<br />
#To the Kan LB a single colony from the transformation plate of [[Melbourne/BBa_I15010|'''BBa_I15010''']] was introduced.<br />
#Cells incubated at 37degrees with shaking overnight.<br />
<br />
===28 June 2007===<br />
<br />
====Miniprep====<br />
#[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:<br />
#*[[Melbourne/pJS010|'''pJS010''']]<br />
#*[[Melbourne/Fusion|'''Fusion''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']]<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']]<br />
#*[[Melbourne/BBa_B0010|'''P2 3P''']]<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']]<br />
#*[[Melbourne/BBa_I15010|'''I15010''']]<br />
#Stored in -20 freezer<br />
<br />
====Digest====<br />
<br />
<br />
====Liquid culture====<br />
#[[Melb:Growing up cells|Cultured]] the following cells from transformed plates:<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)<br />
#*[[Melbourne/BBa_I15010|'''BBa_I15010''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)<br />
#*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)<br />
#*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)<br />
<br />
===29 June 2007===<br />
====Miniprep====<br />
#[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)<br />
#*[[Melbourne/BBa_I15010|'''BBa_I15010''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)<br />
#*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)<br />
#*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)<br />
<br />
#Stored in -20 freezer<br />
<br />
===30 June 2007===<br />
==Week 2==<br />
*2 July 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#Q04510<br />
*#E0241<br />
*#E0040<br />
*#B0014<br />
*#J61035<br />
<br />
*3 July 2007: [[Melb:Transformation Protocol|Re Transformed]] into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.<br />
*#[[Melbourne/BBa_J61035|'''P4 8J''']] -> Three colonies -> grew in liquid culture 4 july<br />
*#[[Melbourne/BBa_E0040|'''P1 5H''']] -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'<br />
*#[[Melbourne/BBa_E0241|'''P2 15L''']] -> Three colonies -> grew in liquid culture 4 july<br />
<br />
*4 July 2007:<br />
<br />
====Ampicillin Plates====<br />
*Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June. <br />
**Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.<br />
<br />
====Tranformation====<br />
[[Melbourne/Transformation Protocol|Transformed]] the following and grew on new ampicillin plates<br />
*[[Melbourne/BBa_E0040|'''P1 5H''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L''']]<br />
**Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)<br />
<br />
====Liquid Culture====<br />
*[[Melbourne/Growing up cells|Cultured]] 2 colonies from each of the following transformed plates and labelled as follows<br />
**[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
**[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
**[[Melbourne/pJS010|'''pJS010 1''']]<br />
**[[Melbourne/pJS010|'''pJS010 2''']]<br />
**[[Melbourne/Fusion|'''Fusion 1''']]<br />
**[[Melbourne/Fusion|'''Fusion 2''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 1''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]<br />
<br />
===5 July 2007===<br />
<br />
====Miniprep====<br />
*1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7<br />
*miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.<br />
**[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
**[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
**[[Melbourne/pJS010|'''pJS010 1''']]<br />
**[[Melbourne/pJS010|'''pJS010 2''']]<br />
**[[Melbourne/Fusion|'''Fusion 1''']]<br />
**[[Melbourne/Fusion|'''Fusion 2''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 1''']](eluted in 130uL due to accidental double application of 50uL elution)<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*the following liquid cultures were not miniprepped due to failure (no growth)<br />
**[[Melbourne/BBa_R0084|'''P1 11H 1''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]<br />
<br />
====Digest====<br />
Performed the following [[Melbourne/Diagnostic Digest|digests]] on DNA from the above miniprep<br />
=====EcoRI/PstI with buffer 3=====<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
*[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
=====EcoRI/HaeII in buffer 2=====<br />
*[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
*[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
*[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
=====XbaI/SpeI in buffer 2=====<br />
*[[Melbourne/pJS010|'''pJS010 1''']]<br />
*[[Melbourne/pJS010|'''pJS010 2''']]<br />
<br />
Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20<br />
<br />
====Transformation====<br />
*[[Melbourne/Transformation Protocol|Transformed]] P1 11H from resuspended DNA.<br />
<br />
====Liquid Culture====<br />
*[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
===6 July 2007===<br />
<br />
====Digest Gel====<br />
* Prepared 20 lane 100mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 0.5xTBE buffer.<br />
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples in the following lane order<br />
*#1kb+ ladder<br />
*#[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
*#[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*#[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
*#[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*#[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
*#[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
*#[[Melbourne/pJS010|'''pJS010 1''']]<br />
*#[[Melbourne/pJS010|'''pJS010 2''']]<br />
*Ran for 1.5hours at 95V<br />
<br />
====Miniprep====<br />
*Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7<br />
*Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.<br />
*[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
<br />
====Digest====<br />
*[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.<br />
*Incubated for 2hours 25min at 37degrees<br />
*Added 5uL 6x loading dye and stored at -20<br />
<br />
====Glycerol Stocks====<br />
The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made:<br />
*Put aside from cultures 5/7 (labelled with this date)<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*Put aside from cultures 6/7 (labelled with this date)<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
Stored at -80<br />
<br />
====Liquid Culture====<br />
[[Melbourne/Growing up cells|Cultured]] the following in 5mL LB<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)<br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)<br />
*[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
<br />
Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7<br />
<br />
===7 July 2007===<br />
<br />
*Made 10x TAE buffer<br />
<br />
====Digest Gel====<br />
*Prepared 8 lane 60mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 1xTAE buffer.<br />
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples from 6/7 in the following lane order.<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
====Glycerol Stocks====<br />
The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made and dated 7/7:<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']] <br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_R0084|'''P1 11H 1''']] <br />
*[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
Stored at -80<br />
<br />
====Miniprep====<br />
*Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.<br />
<br />
====Digest====<br />
*[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA <br />
**EcoRI/PstI in buffer 3<br />
***[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)<br />
***[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
***[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)<br />
***[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
*Incubated for 3hours at 37degrees<br />
*Added 5uL 6x loading dye and stored at -20<br />
<br />
===8 July 2007===<br />
<br />
====Transformation====<br />
Resuspended and [[Melbourne/Transformation Protocol|transformed]] the following<br />
*[[Melbourne/BBa_R0082|'''P1 15P''']](BBa_R0082, Omp R+, Amp)<br />
*[[Melbourne/BBa_R0083|'''P1 17H''']](BBa_R0083, truncated BBa_R0082 Omp R+, Amp)<br />
*[[Melbourne/BBa_E0430|'''P1 11A''']](BBa_E0430, EYFP(RBS+,LVA-,term) Amp)<br />
*[[Melbourne/BBa_E0840|'''P1 16E''']](BBa_E0430; RBS,GFP,term; Amp)<br />
The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.<br />
*[[Melbourne/BBa_Q04510|'''P2 13K''']] (BBa_Q04510, c1 inverter, Kan)<br />
<br />
==Week 3==<br />
===9 July 2007===<br />
*Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run<br />
*liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K<br />
===10 July 2007===<br />
*Miniprep<br />
*Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H 1.0hrs run<br />
*liquid culture I15010, P2<br />
===11 July 2007===<br />
*Digest for ligation P1-15P(1)10/7,P1-11H 10/7,P1-17H(1)10/7,P1-16E(2)10/7,P1-11A(1)10/7<br />
*loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H<br />
** Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.<br />
** Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA<br />
*Excise bands of interest and purify invitrogen<br />
*liquid culture P1-11A,P1-15P 10ml<br />
*Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.<br />
*Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H<br />
===12 July 2007===<br />
*Ran Gel P1-11A,P1-16E,P1-15P,P1-11H<br />
*miniprepped P1-11A,P1-15P<br />
*Digest<br />
*Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)<br />
*Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)<br />
*Liquid culture transformants 11/7<br />
===13 July 2007===<br />
*miniprep cultures from transformants 11/7<br />
*Digestion P1-15P and P1-11A from 12/7/07 37degC 3hours 15 minutes stopped 5uL of 6X loading dye.<br />
*Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.<br />
*Glycerol stocks of P3-20I,P2-21B,P2-23N<br />
*Transform DH5a with ligation product<br />
===14 July 2007===<br />
*Transform using 10uL of ligation reaction<br />
==Week 4==<br />
===16 July 2007===<br />
*[[Melbourne/colony pcr|Colony PCR]]<br />
===17 July 2007===<br />
===18 July 2007===<br />
*Purchased XbaI,EcoRI,PstI<br />
*Miniprepped cultures 1,3,6 from 16/7<br />
*Digestion with E/P<br />
*Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?<br />
<br />
===19 July 2007===<br />
===20 July 2007===<br />
==Week 5==<br />
===23 July 2007===<br />
===24 July 2007===<br />
===25 July 2007===<br />
===26 July 2007===<br />
===27 July 2007===<br />
==Week 6==<br />
===30 July 2007===<br />
===31 July 2007===<br />
===1 Aug 2007===<br />
===2 Aug 2007===<br />
===3 Aug 2007===<br />
===4 Aug 2007===<br />
==Week 7==<br />
===5 Aug 2007===<br />
===6 Aug 2007===<br />
===7 Aug 2007===<br />
===8 Aug 2007===<br />
===9 Aug 2007===<br />
===10 Aug 2007===<br />
===11 Aug 2007===<br />
==Week 8==<br />
===12 Aug 2007===<br />
===13 Aug 2007===<br />
===14 Aug 2007===<br />
===15 Aug 2007===<br />
===16 Aug 2007===<br />
===17 Aug 2007===<br />
===18 Aug 2007===<br />
==Week 9==<br />
===19 Aug 2007===<br />
===20 Aug 2007===<br />
===21 Aug 2007===<br />
===22 Aug 2007===<br />
===23 Aug 2007===<br />
===24 Aug 2007===<br />
===25 Aug 2007===<br />
==Week 10==<br />
===26 Aug 2007===<br />
===27 Aug 2007===<br />
===28 Aug 2007===<br />
===29 Aug 2007===<br />
===30 Aug 2007===<br />
===31 Aug 2007===<br />
===1 Sept 2007===<br />
==Week 11==<br />
===2 Sept 2007===<br />
===3 Sept 2007===<br />
===4 Sept 2007===<br />
===5 Sept 2007===<br />
===6 Sept 2007===<br />
===7 Sept 2007===<br />
===8 Sept 2007===<br />
==Week 12==<br />
===9 Sept 2007===<br />
===10 Sept 2007===<br />
===11 Sept 2007===<br />
===12 Sept 2007===<br />
===13 Sept 2007===<br />
===14 Sept 2007===<br />
===15 Sept 2007===<br />
==Week 13==<br />
===16 Sept 2007===<br />
===17 Sept 2007===<br />
===18 Sept 2007===<br />
===19 Sept 2007===<br />
===20 Sept 2007===<br />
===21 Sept 2007===<br />
===22 Sept 2007===<br />
==Week 13==<br />
===23 Sept 2007===<br />
===24 Sept 2007===<br />
===25 Sept 2007===<br />
===26 Sept 2007===<br />
*Miniprepped the following<br />
**TetR-RBS-P2,21A-ter A,B,C and D<br />
**TetR-RBS-ComP-ter A,B,C and D<br />
**GenRBS-ComA-ter A,B,C and D<br />
**cI-L3 A,B,C and D<br />
<br />
===27 Sept 2007===<br />
===28 Sept 2007===<br />
===29 Sept 2007===<br />
==Week 14==<br />
===30 Sept 2007===<br />
===1 Oct 2007===<br />
===2 Oct 2007===<br />
===3 Oct 2007===<br />
===4 Oct 2007===<br />
===5 Oct 2007===<br />
===6 Oct 2007===<br />
==Week 15==<br />
===7 Oct 2007===<br />
===8 Oct 2007===<br />
===9 Oct 2007===<br />
===10 Oct 2007===<br />
===11 Oct 2007===<br />
===12 Oct 2007===<br />
===13 Oct 2007===<br />
==Week 16==<br />
===14 Oct 2007===<br />
===15 Oct 2007===<br />
===16 Oct 2007===<br />
===17 Oct 2007===<br />
===18 Oct 2007===<br />
===19 Oct 2007===<br />
===20 Oct 2007===<br />
==Week 16==<br />
===21 Oct 2007===<br />
===22 Oct 2007===<br />
===23 Oct 2007===<br />
===24 Oct 2007===<br />
===25 Oct 2007===<br />
===26 Oct 2007===</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook
Melbourne/Lab Notebook
2007-09-27T00:26:55Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<Back to team home page>]] <br />
<br />
==Week 1==<br />
*25 June 2007: Prepared LB agar plates Amp & Kana.<br />
*25 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melbourne/Transformation Protocol shorter|Transformed (shorter protocol)]] into competent DH5alpha cells.<br />
*#[[Melbourne/BBa_I15008|'''P2 21A''']] - (BBa_I15008, ho1, Kan) -> No colonies on plates<br />
*#[[Melbourne/BBa_I15009|'''P2 21C''']] - (BBa_I15009, PcyA, Kan) ->No colonies on plates<br />
*#[[Melbourne/BBa_R0084|'''P1 11H''']] - (BBa_R0084, OmpR positive promoter, Amp)->Small number of colonies.<br />
<br />
<br />
<br />
*26 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034, RBS, Amp)<br />
*#[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051, c1 protein, Amp)<br />
*#[[Melbourne/BBa_B0010|'''P2 3P''']] (BBa_B0010, Terminator, Amp)<br />
<br />
*26 June 2007: Streaked the following cells:<br />
*#[[Melbourne/pJS010|'''pJS010''']] (from solid agar, Amp)<br />
*#[[Melbourne/Fusion|'''Fusion protein''']] (from glycerol stock, Amp?)<br />
*#[[Melbourne/BBa_I15010|'''BBa_I15010''']] (from solid agar, Kan)<br />
<br />
*27 June 2007: [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#[[Melbourne/BBa_I15008|'''P2 21A''']] (Kan)<br />
*#[[Melbourne/BBa_I15009|'''P2 21C''']] (Kan)<br />
<br />
<br />
====Liquid culture====<br />
#[[Melb:Growing up cells|Prepared]] 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).<br />
#Aliquoted 5mL Amp LB into 6 50mL falcon tubes<br />
#To the Amp LB aliquots single transformed colonies from plates of the following were introduced:<br />
#*[[Melbourne/pJS010|'''pJS010''']]<br />
#*[[Melbourne/Fusion|'''Fusion''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']]<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']]<br />
#*[[Melbourne/BBa_B0010|'''P2 3P''']]<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']]<br />
#To the Kan LB a single colony from the transformation plate of [[Melbourne/BBa_I15010|'''BBa_I15010''']] was introduced.<br />
#Cells incubated at 37degrees with shaking overnight.<br />
<br />
===28 June 2007===<br />
<br />
====Miniprep====<br />
#[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:<br />
#*[[Melbourne/pJS010|'''pJS010''']]<br />
#*[[Melbourne/Fusion|'''Fusion''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']]<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']]<br />
#*[[Melbourne/BBa_B0010|'''P2 3P''']]<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']]<br />
#*[[Melbourne/BBa_I15010|'''I15010''']]<br />
#Stored in -20 freezer<br />
<br />
====Digest====<br />
<br />
<br />
====Liquid culture====<br />
#[[Melb:Growing up cells|Cultured]] the following cells from transformed plates:<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)<br />
#*[[Melbourne/BBa_I15010|'''BBa_I15010''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)<br />
#*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)<br />
#*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)<br />
<br />
===29 June 2007===<br />
====Miniprep====<br />
#[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:<br />
#*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)<br />
#*[[Melbourne/BBa_I15010|'''BBa_I15010''']]<br />
#*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)<br />
#*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)<br />
#*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)<br />
#*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)<br />
<br />
#Stored in -20 freezer<br />
<br />
===30 June 2007===<br />
==Week 2==<br />
*2 July 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.<br />
*#Q04510<br />
*#E0241<br />
*#E0040<br />
*#B0014<br />
*#J61035<br />
<br />
*3 July 2007: [[Melb:Transformation Protocol|Re Transformed]] into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.<br />
*#[[Melbourne/BBa_J61035|'''P4 8J''']] -> Three colonies -> grew in liquid culture 4 july<br />
*#[[Melbourne/BBa_E0040|'''P1 5H''']] -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'<br />
*#[[Melbourne/BBa_E0241|'''P2 15L''']] -> Three colonies -> grew in liquid culture 4 july<br />
<br />
*4 July 2007:<br />
<br />
====Ampicillin Plates====<br />
*Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June. <br />
**Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.<br />
<br />
====Tranformation====<br />
[[Melbourne/Transformation Protocol|Transformed]] the following and grew on new ampicillin plates<br />
*[[Melbourne/BBa_E0040|'''P1 5H''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L''']]<br />
**Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)<br />
<br />
====Liquid Culture====<br />
*[[Melbourne/Growing up cells|Cultured]] 2 colonies from each of the following transformed plates and labelled as follows<br />
**[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
**[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
**[[Melbourne/pJS010|'''pJS010 1''']]<br />
**[[Melbourne/pJS010|'''pJS010 2''']]<br />
**[[Melbourne/Fusion|'''Fusion 1''']]<br />
**[[Melbourne/Fusion|'''Fusion 2''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 1''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]<br />
<br />
===5 July 2007===<br />
<br />
====Miniprep====<br />
*1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7<br />
*miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.<br />
**[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
**[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
**[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
**[[Melbourne/pJS010|'''pJS010 1''']]<br />
**[[Melbourne/pJS010|'''pJS010 2''']]<br />
**[[Melbourne/Fusion|'''Fusion 1''']]<br />
**[[Melbourne/Fusion|'''Fusion 2''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
**[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
**[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 1''']](eluted in 130uL due to accidental double application of 50uL elution)<br />
**[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
**[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*the following liquid cultures were not miniprepped due to failure (no growth)<br />
**[[Melbourne/BBa_R0084|'''P1 11H 1''']]<br />
**[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
**[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]<br />
<br />
====Digest====<br />
Performed the following [[Melbourne/Diagnostic Digest|digests]] on DNA from the above miniprep<br />
=====EcoRI/PstI with buffer 3=====<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
*[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
=====EcoRI/HaeII in buffer 2=====<br />
*[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
*[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
*[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
=====XbaI/SpeI in buffer 2=====<br />
*[[Melbourne/pJS010|'''pJS010 1''']]<br />
*[[Melbourne/pJS010|'''pJS010 2''']]<br />
<br />
Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20<br />
<br />
====Transformation====<br />
*[[Melbourne/Transformation Protocol|Transformed]] P1 11H from resuspended DNA.<br />
<br />
====Liquid Culture====<br />
*[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
===6 July 2007===<br />
<br />
====Digest Gel====<br />
* Prepared 20 lane 100mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 0.5xTBE buffer.<br />
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples in the following lane order<br />
*#1kb+ ladder<br />
*#[[Melbourne/BBa_I15010|'''I15010 1''']]<br />
*#[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*#[[Melbourne/BBa_Q04510|'''P2 13K 1''']]<br />
*#[[Melbourne/BBa_Q04510|'''P2 13K 2''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*#[[Melbourne/BBa_B0010|'''P2 3P 1''']]<br />
*#[[Melbourne/BBa_B0010|'''P2 3P 2''']]<br />
*#[[Melbourne/pJS010|'''pJS010 1''']]<br />
*#[[Melbourne/pJS010|'''pJS010 2''']]<br />
*Ran for 1.5hours at 95V<br />
<br />
====Miniprep====<br />
*Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7<br />
*Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.<br />
*[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
<br />
====Digest====<br />
*[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.<br />
*Incubated for 2hours 25min at 37degrees<br />
*Added 5uL 6x loading dye and stored at -20<br />
<br />
====Glycerol Stocks====<br />
The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made:<br />
*Put aside from cultures 5/7 (labelled with this date)<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 1''']]<br />
*#[[Melbourne/BBa_B0014|'''P1 1G 2''']]<br />
*Put aside from cultures 6/7 (labelled with this date)<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
Stored at -80<br />
<br />
====Liquid Culture====<br />
[[Melbourne/Growing up cells|Cultured]] the following in 5mL LB<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)<br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)<br />
*[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
<br />
Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7<br />
<br />
===7 July 2007===<br />
<br />
*Made 10x TAE buffer<br />
<br />
====Digest Gel====<br />
*Prepared 8 lane 60mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 1xTAE buffer.<br />
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples from 6/7 in the following lane order.<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 1''']]<br />
*#[[Melbourne/BBa_J61035|'''P4 8J 2''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 1''']]<br />
*#[[Melbourne/BBa_E0040|'''P1 5H 2''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 1''']]<br />
*#[[Melbourne/BBa_E0241|'''P2 15L 2''']]<br />
<br />
====Glycerol Stocks====<br />
The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made and dated 7/7:<br />
*[[Melbourne/BBa_I15010|'''I15010 1''']] <br />
*[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
*[[Melbourne/BBa_R0084|'''P1 11H 1''']] <br />
*[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
Stored at -80<br />
<br />
====Miniprep====<br />
*Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.<br />
<br />
====Digest====<br />
*[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA <br />
**EcoRI/PstI in buffer 3<br />
***[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)<br />
***[[Melbourne/BBa_I15010|'''I15010 2''']]<br />
***[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)<br />
***[[Melbourne/BBa_R0084|'''P1 11H 2''']]<br />
*Incubated for 3hours at 37degrees<br />
*Added 5uL 6x loading dye and stored at -20<br />
<br />
===8 July 2007===<br />
<br />
====Transformation====<br />
Resuspended and [[Melbourne/Transformation Protocol|transformed]] the following<br />
*[[Melbourne/BBa_R0082|'''P1 15P''']](BBa_R0082, Omp R+, Amp)<br />
*[[Melbourne/BBa_R0083|'''P1 17H''']](BBa_R0083, truncated BBa_R0082 Omp R+, Amp)<br />
*[[Melbourne/BBa_E0430|'''P1 11A''']](BBa_E0430, EYFP(RBS+,LVA-,term) Amp)<br />
*[[Melbourne/BBa_E0840|'''P1 16E''']](BBa_E0430; RBS,GFP,term; Amp)<br />
The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.<br />
*[[Melbourne/BBa_Q04510|'''P2 13K''']] (BBa_Q04510, c1 inverter, Kan)<br />
<br />
==Week 3==<br />
===9 July 2007===<br />
*Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run<br />
*liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K<br />
===10 July 2007===<br />
*Miniprep<br />
*Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H 1.0hrs run<br />
*liquid culture I15010, P2<br />
===11 July 2007===<br />
*Digest for ligation P1-15P(1)10/7,P1-11H 10/7,P1-17H(1)10/7,P1-16E(2)10/7,P1-11A(1)10/7<br />
*loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H<br />
** Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.<br />
** Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA<br />
*Excise bands of interest and purify invitrogen<br />
*liquid culture P1-11A,P1-15P 10ml<br />
*Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.<br />
*Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H<br />
===12 July 2007===<br />
*Ran Gel P1-11A,P1-16E,P1-15P,P1-11H<br />
*miniprepped P1-11A,P1-15P<br />
*Digest<br />
*Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)<br />
*Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)<br />
*Liquid culture transformants 11/7<br />
===13 July 2007===<br />
*miniprep cultures from transformants 11/7<br />
*Digestion P1-15P and P1-11A from 12/7/07 37degC 3hours 15 minutes stopped 5uL of 6X loading dye.<br />
*Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.<br />
*Glycerol stocks of P3-20I,P2-21B,P2-23N<br />
*Transform DH5a with ligation product<br />
===14 July 2007===<br />
*Transform using 10uL of ligation reaction<br />
==Week 4==<br />
===16 July 2007===<br />
*[[Melbourne/colony pcr|Colony PCR]]<br />
===17 July 2007===<br />
===18 July 2007===<br />
*Purchased XbaI,EcoRI,PstI<br />
*Miniprepped cultures 1,3,6 from 16/7<br />
*Digestion with E/P<br />
*Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?<br />
<br />
===19 July 2007===<br />
===20 July 2007===<br />
==Week 5==<br />
===23 July 2007===<br />
===24 July 2007===<br />
===25 July 2007===<br />
===26 July 2007===<br />
===27 July 2007===<br />
==Week 6==<br />
===30 July 2007===<br />
===31 July 2007===<br />
===1 Aug 2007===<br />
===2 Aug 2007===<br />
===3 Aug 2007===<br />
===4 Aug 2007===<br />
==Week 7==<br />
===5 Aug 2007===<br />
===6 Aug 2007===<br />
===7 Aug 2007===<br />
===8 Aug 2007===<br />
===9 Aug 2007===<br />
===10 Aug 2007===<br />
===11 Aug 2007===<br />
==Week 8==<br />
===12 Aug 2007===<br />
===13 Aug 2007===<br />
===14 Aug 2007===<br />
===15 Aug 2007===<br />
===16 Aug 2007===<br />
===17 Aug 2007===<br />
===18 Aug 2007===<br />
==Week 9==<br />
===19 Aug 2007===<br />
===20 Aug 2007===<br />
===21 Aug 2007===<br />
===22 Aug 2007===<br />
===23 Aug 2007===<br />
===24 Aug 2007===<br />
===25 Aug 2007===<br />
==Week 10==<br />
===26 Aug 2007===<br />
===27 Aug 2007===<br />
===28 Aug 2007===<br />
===29 Aug 2007===<br />
===30 Aug 2007===<br />
===31 Aug 2007===<br />
===1 Sept 2007===<br />
==Week 11==<br />
===2 Sept 2007===<br />
===3 Sept 2007===<br />
===4 Sept 2007===<br />
===5 Sept 2007===<br />
===6 Sept 2007===<br />
===7 Sept 2007===<br />
===8 Sept 2007===<br />
==Week 12==<br />
===9 Sept 2007===<br />
===10 Sept 2007===<br />
===11 Sept 2007===<br />
===12 Sept 2007===<br />
===13 Sept 2007===<br />
===14 Sept 2007===<br />
===15 Sept 2007===<br />
==Week 13==<br />
===16 Sept 2007===<br />
===17 Sept 2007===<br />
===18 Sept 2007===<br />
===19 Sept 2007===<br />
===20 Sept 2007===<br />
===21 Sept 2007===<br />
===22 Sept 2007===<br />
==Week 13==<br />
===23 Sept 2007===<br />
===24 Sept 2007===<br />
===25 Sept 2007===<br />
===26 Sept 2007===<br />
===27 Sept 2007===<br />
===28 Sept 2007===<br />
===29 Sept 2007===<br />
==Week 14==<br />
===30 Sept 2007===<br />
===1 Oct 2007===<br />
===2 Oct 2007===<br />
===3 Oct 2007===<br />
===4 Oct 2007===<br />
===5 Oct 2007===<br />
===6 Oct 2007===<br />
==Week 15==<br />
===7 Oct 2007===<br />
===8 Oct 2007===<br />
===9 Oct 2007===<br />
===10 Oct 2007===<br />
===11 Oct 2007===<br />
===12 Oct 2007===<br />
===13 Oct 2007===<br />
==Week 16==<br />
===14 Oct 2007===<br />
===15 Oct 2007===<br />
===16 Oct 2007===<br />
===17 Oct 2007===<br />
===18 Oct 2007===<br />
===19 Oct 2007===<br />
===20 Oct 2007===<br />
==Week 16==<br />
===21 Oct 2007===<br />
===22 Oct 2007===<br />
===23 Oct 2007===<br />
===24 Oct 2007===<br />
===25 Oct 2007===<br />
===26 Oct 2007===</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/meeting_24_August_2007
Melbourne/meeting 24 August 2007
2007-08-25T00:29:12Z
<p>Patricia: </p>
<hr />
<div>== 5:30 Action points from last meeting:==<br />
<br />
*Pat & Alisa: LacZ reporter plan - complete<br />
<br />
*Jan: Plan for blue light on wiki by 17th August- Done but needs more work: couldn’t get anyone else to implement it based on this!<br />
<br />
*Phil: re prism :Edmund optics:VIS60 Schott veril linear variable interference filter SGD$1600 25mm wide 60mm long linear 400nm-700nm (contact sheet)<br />
N47-279 eqilateral prism 35mm long 35mm face SGD$162<br />
<br />
*Phil: Chase Status of #BBa-M30109 & #BBa-V1012 - Arrived<br />
<br />
*Craig: Alkaline lysis technique for miniprep to be documented<br />
<br />
*Craig: Protein extraction technique –done sent to Phil by email.<br />
<br />
*Cheng: Daisy to produce competent cells of DB3.1 and Enz- strains: Alisa will set up plates for Daisy to work from by monday so we will have a store of competant cells by Tues/Wed<br />
<br />
*Chris: Gas vesicle expression experiment by 21/8/07.-Done<br />
<br />
<br />
<br />
== 5:35 Status Reports: (5 minutes each including any discussion)==<br />
<br />
*Alisa: Ligation experiments progress <br />
**Craig ligated the ompR promoter to the RBS-LacZ-Ter and c1 inverter, confirmation digest and gel to be done on saturday and parts sent for sequencing confirmation on Monday. <br />
**Additional ligations to be set up and confirmed during the week: OmpR test harness(Ompr-RBS-LacZ-ter) to be ligated behind M30109, Ompr-c1 inverter to be ligated behind M30109.<br />
<br />
*Pat & Alisa: Red light receptor progress/design/plan- needs?<br />
**these include to above ligations. We need to work out how we are going to test these parts<br />
<br />
*Jan: Blue light receptor design/plan progress - needs? (Absent from meeting)<br />
<br />
*Phil: Gas vesicles progress/design/plan - needs?<br />
**Progress good, now on round 3 of mutagenesis (3 plates x2).<br />
**Need miniprep of six colonies on Monday and Digest with PstI, I will pick colonies Sunday.<br />
**Buoyancy experiment confused re IPTG, -IPTG,+IPTG but definitely differences. See attached photos at 1hour,6hours,18hours,96hours and last 3 at 126hours<br />
**Other parts required from kit->glycerol stocks: B034 (P1-3O) , B0015 (P1-1I or P3-3O), J61034 (P4-8F or 8H)<br />
<br />
<br />
==5.55 New Agenda Items:==<br />
<br />
*Phil: Administrative Timetable: (8 Weeks to go)<br />
<br />
*Final Team roster and Jamboree attendance fees due. Who is going? Jan & Phil perhaps, others??? Need to find out state of funds so that we can work out how many people we are ''able'' to send<br />
*Chris: Next years Comp Plan/preparations - same state as last week<br />
<br />
*Any other business<br />
<br />
* Next meeting: <b/> 28th August (Friday) 5.30PM.<br />
<br />
==6:15 End ==<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Igem Administrative Timetable:==<br />
<br />
{|border="1"<br />
<br />
|1 September || Final team rosters due. <br />
<br />
|-<br />
<br />
|1 September|| Jamboree attendance fees due $100USD per team member. <br />
<br />
|-<br />
<br />
|26 October|| Project and part documentation due DNA received USA.<br />
<br />
|-<br />
<br />
|26 October || BioBrick Part DNA received by the Registry. <br />
<br />
|-<br />
<br />
|3-4 November || iGEM Competition Jamboree, MIT, USA. <br />
<br />
|}<br />
<br />
<br />
<br />
.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Documentation wiki status:==<br />
<br />
**Priority should be on plans and experimental report analysis and conclusions-----Alisa .<br />
<br />
**If only Pat & Alisa doing lab work not much documentation needed.<br />
<br />
**Equipement:<br />
<br />
*** MissingLas3000 method (in lab notebook)<br />
<br />
*** Missing DNA measurement protocol (eppendorf)<br />
<br />
**Reagents pages incomplete: <br />
<br />
***Some location pictures not finished or uploaded.<br />
<br />
*** Some newer reagents not entered.<br />
<br />
**Secondary reagents pages not made:<br />
<br />
*** LB, Plates, TAE , NZY+ Broth<br />
<br />
**Protocol part:<br />
<br />
*** No connections from Protocols to reagents.<br />
<br />
*** Ligation protocol missing<br />
<br />
**Lab notebook:<br />
<br />
*** Format uses too many lines to see connections between days.<br />
<br />
*** Latter part missing links<br />
<br />
*** Can table of contents be restricted to date level?<br />
<br />
**Experimental reports:<br />
<br />
*** Lacks discussion/conclusion<br />
<br />
*** Ligation experiment missing<br />
<br />
*** Lattest few gells not marked up and entered.<br />
<br />
**Project overview link broken<br />
<br />
**Background: <br />
<br />
*** GFP missing , <br />
<br />
*** Red photo sensor to GFP test harness missing.<br />
<br />
**Plan: Only Gas vesicles plan entered.<br />
<br />
<br />
<br />
{|border="1"<br />
<br />
!Individual!!Desired time committment<br />
<br />
|-<br />
<br />
|Pat|| About 4h/wk Monday afternoon or Tuesday morning<br />
<br />
|-<br />
<br />
|Alisa|| About 4h/wk - Thursday afternoon<br />
<br />
|-<br />
<br />
|Jan || About 10h/pw - flexible weekday mornings<br />
<br />
|-<br />
<br />
|Phil || About 18h/pw - Thursday morning, Friday, Saturday or Sunday, <br />
<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/meeting_minutes
Melbourne/meeting minutes
2007-08-25T00:19:09Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<Back to team home page>]] [[Melbourne/meeting minutes:T|<Template>]]<br />
*[[Melbourne/meeting 13 april 2007]]<br />
*[[Melbourne/meeting 20 April 2007]]<br />
*[[Melbourne/meeting 30 June 2007]]<br />
*[[Melbourne/meeting 19 July 2007]]<br />
*[[Melbourne/meeting 27 July 2007]]<br />
*[[Melbourne/meeting 3 August 2007]]<br />
*[[Melbourne/meeting 10 August 2007]]<br />
*[[Melbourne/meeting 17 August 2007]]<br />
*[[Melbourne/meeting 24 August 2007]]</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_A
Melbourne/BBa P1010 A
2007-08-17T07:36:11Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:<br />
<br />
==Physical DNA==<br />
*Registry: BBa_<br />
*Kit location: <br />
*Resistance: <br />
*Part Size:<br />
*Plasmid Vector:<br />
*Vector Size:<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: <br />
***Location: <br />
**Confirmed: <br />
**Glycerol Stock: <br />
***Location:<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_AK
Melbourne/BBa P1010 AK
2007-08-17T07:35:55Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:<br />
<br />
==Physical DNA==<br />
*Registry: BBa_<br />
*Kit location: <br />
*Resistance: <br />
*Part Size:<br />
*Plasmid Vector:<br />
*Vector Size:<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: <br />
***Location: <br />
**Confirmed: <br />
**Glycerol Stock: <br />
***Location:<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_P1010_AC
Melbourne/BBa P1010 AC
2007-08-17T07:35:37Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:<br />
<br />
==Physical DNA==<br />
*Registry: BBa_<br />
*Kit location: <br />
*Resistance: <br />
*Part Size:<br />
*Plasmid Vector:<br />
*Vector Size:<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: <br />
***Location: <br />
**Confirmed: <br />
**Glycerol Stock: <br />
***Location:<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/BBa_E0033
Melbourne/BBa E0033
2007-08-17T07:35:20Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:<br />
<br />
==Physical DNA==<br />
*Registry: BBa_<br />
*Kit location: <br />
*Resistance: <br />
*Part Size:<br />
*Plasmid Vector:<br />
*Vector Size:<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: <br />
***Location: <br />
**Confirmed: <br />
**Glycerol Stock: <br />
***Location:<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Part_template
Melbourne/Part template
2007-08-17T07:34:31Z
<p>Patricia: /* Expected Digestion Products */</p>
<hr />
<div>[[Melbourne/Parts used|<Return to Parts Used>]] [[Melbourne/Lab Notebook|<Return to Lab notebook>]] [[Melbourne|<Return to team homepage>]]<br />
=Name:=<br />
Last updated:<br />
<br />
==Physical DNA==<br />
*Registry: BBa_<br />
*Kit location: <br />
*Resistance: <br />
*Part Size:<br />
*Plasmid Vector:<br />
*Vector Size:<br />
<br />
==Status==<br />
*Labelling:<br />
*Resuspended:<br />
**Location: <br />
*Transformed: <br />
**Location: <br />
**Miniprepped: <br />
***Location: <br />
**Confirmed: <br />
**Glycerol Stock: <br />
***Location:<br />
<br />
<br />
==Expected Digestion Products==<br />
<br />
{| border="1"<br />
|+ Expected Digestion Products<br />
! Digest !! Fragment 1 !! Fragment 2<br />
|-<br />
! EcoRI/SpeI<br />
| || <br />
|-<br />
! EcoRI/HaeII<br />
| || <br />
|}<br />
<br />
<br />
<br />
__NOTOC__</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_used
Melbourne/Parts used
2007-08-17T07:32:31Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: 17/8/07<br />
{| border="1"<br />
|+ Parts Used by Melbourne [[Melbourne/parts used graphical| <GRAPHICAL>]]<br />
! Label !! Registry Part Number !! Part Name !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status<br />
|-<br />
! [[Melbourne/BBa_B0014|P1 1G]]<br />
| BBa_B0014 || Double Terminator || 95 || pSB1AK3 || 3189 || 3284 || Amp, Kan || Glycerol stocks in -80<br />
|-<br />
! [[Melbourne/BBa_B0034|P1 3O]]<br />
| BBa_B0034 || RBS || 12 || pSB1A2 || 2079 || 2091 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_C0051|P1 5G]]<br />
| BBa_C0051 || c1 represser protein || 750 || pSB1A2 || 2079 || 2892 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0040|P1 5H]]<br />
| BBa_E0040 || GFP || 720 || pSB1A2 || 2079 || 2799 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0430|P1 11A]]<br />
| BBa_E0430 || EYFP(RBS+,LVA-,term) || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0084|P1 11H]]<br />
| BBa_R0084 || OmpR Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0082|P1 15P]]<br />
| BBa_R0082 || OmpR+ Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0840|P1 16E]]<br />
| BBa_E0840 || RBS,GFP,term || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0083|P1 17H]]<br />
| BBa_R0083 || OmpR+ Promoter(truncated version of R0082 || 79 || pSB1A2 || 2079 || 2157 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_B0010|P2 3P]]<br />
| BBa_B0010 || Single Terminator || 80 || pSB1A2 || 2079 || 2159 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0033|P2 9E]]<br />
| BBa_E0033 || LacZ alpha || 353 || pSB2K3 || 4425 || 4778 || Kan || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_Q04510|P2 13K]]<br />
| BBa_Q04510 || c1 inverter || 987 || pSB2K3 || 4425 || 5412 || Kan || Transformed<br />
|-<br />
! [[Melbourne/BBa_E0241|P2 15L]]<br />
| BBa_E0241 || PoPs -> GFP || 795 || pSB1A2 || 2079 || 2874 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15008|P2 21A]]<br />
| BBa_I15008 || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AC|P2 21B]]<br />
| BBa_P1010 || Death Plasmid AmpR/ChlR || 675 || pSB1AC3 || 3055 || 3730 || A/C || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15009|P2 21C]]<br />
| BBa_I15009 || PcyA || 750 || pSB2K3 || 4425 || 5175 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_AK|P2 23N]]<br />
| BBa_P1010 || Death Plasmid AmpR/KanR || 675 || pSB1AK3 || 3189 || 3864 || Amp.Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_P1010_A|P3 20I]]<br />
| BBa_P1010 || Death Plasmid AmpR || 675 || pSB1A3 || 2157 || 2832 || Kan || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_J61035|P4 8J]]<br />
| BBa_J61035 || EcoRI-GenR-XbaI || 3551 || J61035 || 3551 || 3551 || Amp, Gen || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15010|I15010]]<br />
| BBa_I15010 || cph8(cph1/Envz fusion || 2238 || pSB2K3 || 4425 || 6663 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/pJS010|pJS010]]<br />
| - || SopII-Htr || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/Fusion|Fusion]]<br />
| - || NpHtrII fusion || || || || || Amp || Glycerol stock in -80<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_used
Melbourne/Parts used
2007-08-17T07:16:12Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: 17/8/07<br />
{| border="1"<br />
|+ Parts Used by Melbourne [[Melbourne/parts used graphical| <GRAPHICAL>]]<br />
! Label !! Registry Part Number !! Part Name !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status<br />
|-<br />
! [[Melbourne/BBa_B0014|P1 1G]]<br />
| BBa_B0014 || Double Terminator || 95 || pSB1AK3 || 3189 || 3284 || Amp, Kan || Glycerol stocks in -80<br />
|-<br />
! [[Melbourne/BBa_B0034|P1 3O]]<br />
| BBa_B0034 || RBS || 12 || pSB1A2 || 2079 || 2091 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_C0051|P1 5G]]<br />
| BBa_C0051 || c1 represser protein || 750 || pSB1A2 || 2079 || 2892 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0040|P1 5H]]<br />
| BBa_E0040 || GFP || 720 || pSB1A2 || 2079 || 2799 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0430|P1 11A]]<br />
| BBa_E0430 || EYFP(RBS+,LVA-,term) || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0084|P1 11H]]<br />
| BBa_R0084 || OmpR Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0082|P1 15P]]<br />
| BBa_R0082 || OmpR+ Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0840|P1 16E]]<br />
| BBa_E0840 || RBS,GFP,term || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0083|P1 17H]]<br />
| BBa_R0083 || OmpR+ Promoter(truncated version of R0082 || 79 || pSB1A2 || 2079 || 2157 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_B0010|P2 3P]]<br />
| BBa_B0010 || Single Terminator || 80 || pSB1A2 || 2079 || 2159 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_Q04510|P2 13K]]<br />
| BBa_Q04510 || c1 inverter || 987 || pSB2K3 || 4425 || 5412 || Kan || Transformed<br />
|-<br />
! [[Melbourne/BBa_E0241|P2 15L]]<br />
| BBa_E0241 || PoPs -> GFP || 795 || pSB1A2 || 2079 || 2874 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15008|P2 21A]]<br />
| BBa_I15008 || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_|P2 21B]]<br />
| BBa_I15008 || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15009|P2 21C]]<br />
| BBa_I15009 || PcyA || 750 || pSB2K3 || 4425 || 5175 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_J61035|P4 8J]]<br />
| BBa_J61035 || EcoRI-GenR-XbaI || 3551 || J61035 || 3551 || 3551 || Amp, Gen || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15010|I15010]]<br />
| BBa_I15010 || cph8(cph1/Envz fusion || 2238 || pSB2K3 || 4425 || 6663 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/pJS010|pJS010]]<br />
| - || SopII-Htr || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/Fusion|Fusion]]<br />
| - || NpHtrII fusion || || || || || Amp || Glycerol stock in -80<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melbourne/Parts_used
Melbourne/Parts used
2007-08-17T07:13:47Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne|<back to team home page>]] [[Melbourne/Part template|<Template>]]<br />
<br />
Last Updated: 10/7/07<br />
{| border="1"<br />
|+ Parts Used by Melbourne [[Melbourne/parts used graphical| <GRAPHICAL>]]<br />
! Label !! Registry Part Number !! Part Name !! Part Size(bp) !! Plasmid Vector !! Plasmid Size(bp) !! Total size(bp) !! Resistance !! Status<br />
|-<br />
! [[Melbourne/BBa_B0014|P1 1G]]<br />
| BBa_B0014 || Double Terminator || 95 || pSB1AK3 || 3189 || 3284 || Amp, Kan || Glycerol stocks in -80<br />
|-<br />
! [[Melbourne/BBa_B0034|P1 3O]]<br />
| BBa_B0034 || RBS || 12 || pSB1A2 || 2079 || 2091 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_C0051|P1 5G]]<br />
| BBa_C0051 || c1 represser protein || 750 || pSB1A2 || 2079 || 2892 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_E0040|P1 5H]]<br />
| BBa_E0040 || GFP || 720 || pSB1A2 || 2079 || 2799 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0430|P1 11A]]<br />
| BBa_E0430 || EYFP(RBS+,LVA-,term) || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0084|P1 11H]]<br />
| BBa_R0084 || OmpR Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0082|P1 15P]]<br />
| BBa_R0082 || OmpR+ Promoter || 108 || pSB1A2 || 2079 || 2187 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_E0840|P1 16E]]<br />
| BBa_E0840 || RBS,GFP,term || 878 || pSB1A2 || 2079 || 2957 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_R0083|P1 17H]]<br />
| BBa_R0083 || OmpR+ Promoter(truncated version of R0082 || 79 || pSB1A2 || 2079 || 2157 || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_B0010|P2 3P]]<br />
| BBa_B0010 || Single Terminator || 80 || pSB1A2 || 2079 || 2159 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_Q04510|P2 13K]]<br />
| BBa_Q04510 || c1 inverter || 987 || pSB2K3 || 4425 || 5412 || Kan || Transformed<br />
|-<br />
! [[Melbourne/BBa_E0241|P2 15L]]<br />
| BBa_E0241 || PoPs -> GFP || 795 || pSB1A2 || 2079 || 2874 || Amp || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15008|P2 21A]]<br />
| BBa_I15008 || ho1 || 726 || pSB2K3 || 4425 || 5151 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_I15009|P2 21C]]<br />
| BBa_I15009 || PcyA || 750 || pSB2K3 || 4425 || 5175 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/BBa_J61035|P4 8J]]<br />
| BBa_J61035 || EcoRI-GenR-XbaI || 3551 || J61035 || 3551 || 3551 || Amp, Gen || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/BBa_I15010|I15010]]<br />
| BBa_I15010 || cph8(cph1/Envz fusion || 2238 || pSB2K3 || 4425 || 6663 || Kan || Discontinued<br />
|-<br />
! [[Melbourne/pJS010|pJS010]]<br />
| - || SopII-Htr || || || || || Amp || Glycerol stock in -80<br />
|-<br />
! [[Melbourne/Fusion|Fusion]]<br />
| - || NpHtrII fusion || || || || || Amp || Glycerol stock in -80<br />
|}</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:AND_Gate
Melb:Plan:AND Gate
2007-08-11T08:08:57Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Plan|<return to plans page>]]<br />
<br />
#Retransform c1 inverter (afternoon + overnight)<br />
#Liquid culture (overnight)<br />
#Isolate plasmid (mini prep kit not working - to big? - best way to isolate?) (half day)<br />
#Confirm Isolation and part using XbaI/PstI digest (half day)<br />
#*If feeling optimistic digest OmpR promoter simultaneously with SpeI/PstI and prepare to gel purify fragments of OmpR promoter (vector) and the inverter(insert)<br />
#If positive ligate (overnight)<br />
#Transform and liquid culture several colonies to confirm (1 afternoon + overnight + 15min + overnight) <br />
#Miniprep and digest to confirm and glycerol stocks. (1 day)<br />
<br />
Next need to ligate to RBS-ComA-Ter construct.</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:AND_Gate
Melb:Plan:AND Gate
2007-08-11T08:07:17Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Plan|<return to plans page>]]<br />
<br />
#Retransform c1 inverter (afternoon + overnight)<br />
#Liquid culture (overnight)<br />
#Isolate plasmid (mini prep kit not working - to big? - best way to isolate?) (half day)<br />
#Confirm Isolation and part using XbaI/PstI digest (half day)<br />
#*If feeling optimistic digest OmpR promoter simultaneously with SpeI/PstI and prepare to gel purify fragments of OmpR promoter (vector) and the inverter(insert)<br />
#If positive ligate (overnight)<br />
#Transform and liquid culture several colonies to confirm (1 afternoon + overnight + 15min + overnight) <br />
#Miniprep and digest to confirm and glycerol stocks.</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:AND_Gate
Melb:Plan:AND Gate
2007-08-11T07:58:40Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Plan|<return to plans page>]]<br />
<br />
#Retransform c1 inverter (afternoon + overnight)<br />
#Liquid culture (overnight)<br />
#Isolate plasmid (mini prep kit not working - to big? - best way to isolate?)<br />
#</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:GFP_Fluorescent_reporter
Melb:Plan:GFP Fluorescent reporter
2007-08-11T07:55:02Z
<p>Patricia: </p>
<hr />
<div>[[Melbourne/Plan|<back to plans>]]<br />
<br />
==LacZ reporter==<br />
#Ligate LacZ coding region to gentamycin RBS part. (Done?)(1 day)<br />
#Transform and select for appropriate construct using Kan/Gen plate. (Done?)(1 day + overnight)<br />
#Liquid culture, miniprep and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (overnight + 1 day)<br />
#Make glycerol stock of RBS-LacZ containing cells. (same day)<br />
#Ligate the RBS-LacZ construct into double terminator plasmid. (same day)<br />
#Transform ligation and select for construct using Gen/Amp plate. (1 day + overnight)<br />
#Liquid culture, miniprep and confirm using EcoRI/Pst digest (overnight + 1 day)<br />
#prepare glycerol stock (same day)<br />
<br />
<br />
This construct can then be ligated to appropriate promoters for use as a test harness with the substrate 3,4-cyclohexenoesculetin--D-galactopyranoside (S-Gal). For information on conditions see supplementary info from the following paper:<br />
<br />
Nature 438, 441-442 (24 November 2005)<br />
Synthetic biology: Engineering Escherichia coli to see light<br />
Anselm Levskaya1, Aaron A. Chevalier2, Jeffrey J. Tabor2, Zachary Booth Simpson2, Laura A. Lavery2, Matthew Levy2, Eric A. Davidson2, Alexander Scouras2, Andrew D. Ellington2,3, Edward M. Marcotte2,3 and Christopher A. Voigt1,4,5<br />
<br />
<br />
Test harnesses needed:<br />
*OmpR promoter: Ligate to promoter (3 days) and express in Envz+ cells under osmotic stress (time?)<br />
*Red light system: Above construct in Envz- cells with red light system<br />
*Blue light system: Ligate to Promoter responding to ComA (3 days), in Envz- cells constitutively expressing ComA and Blue light receptors (time?)<br />
*AND gate: Use above construct in Envz- cells constitutively expressing Blue light receptor and Red light receptor with ComA expression under the control of OmpR promoter. (time?)</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:GFP_Fluorescent_reporter
Melb:Plan:GFP Fluorescent reporter
2007-08-11T07:53:52Z
<p>Patricia: /* LacZ reporter */</p>
<hr />
<div>==LacZ reporter==<br />
#Ligate LacZ coding region to gentamycin RBS part. (Done?)(1 day)<br />
#Transform and select for appropriate construct using Kan/Gen plate. (Done?)(1 day + overnight)<br />
#Liquid culture, miniprep and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (overnight + 1 day)<br />
#Make glycerol stock of RBS-LacZ containing cells. (same day)<br />
#Ligate the RBS-LacZ construct into double terminator plasmid. (same day)<br />
#Transform ligation and select for construct using Gen/Amp plate. (1 day + overnight)<br />
#Liquid culture, miniprep and confirm using EcoRI/Pst digest (overnight + 1 day)<br />
#prepare glycerol stock (same day)<br />
<br />
<br />
This construct can then be ligated to appropriate promoters for use as a test harness with the substrate 3,4-cyclohexenoesculetin--D-galactopyranoside (S-Gal). For information on conditions see supplementary info from the following paper:<br />
<br />
Nature 438, 441-442 (24 November 2005)<br />
Synthetic biology: Engineering Escherichia coli to see light<br />
Anselm Levskaya1, Aaron A. Chevalier2, Jeffrey J. Tabor2, Zachary Booth Simpson2, Laura A. Lavery2, Matthew Levy2, Eric A. Davidson2, Alexander Scouras2, Andrew D. Ellington2,3, Edward M. Marcotte2,3 and Christopher A. Voigt1,4,5<br />
<br />
<br />
Test harnesses needed:<br />
*OmpR promoter: Ligate to promoter (3 days) and express in Envz+ cells under osmotic stress (time?)<br />
*Red light system: Above construct in Envz- cells with red light system<br />
*Blue light system: Ligate to Promoter responding to ComA (3 days), in Envz- cells constitutively expressing ComA and Blue light receptors (time?)<br />
*AND gate: Use above construct in Envz- cells constitutively expressing Blue light receptor and Red light receptor with ComA expression under the control of OmpR promoter. (time?)</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:GFP_Fluorescent_reporter
Melb:Plan:GFP Fluorescent reporter
2007-08-11T07:53:37Z
<p>Patricia: </p>
<hr />
<div>==LacZ reporter==<br />
#Ligate LacZ coding region to gentamycin RBS part. (Done?)(1 day)<br />
#Transform and select for appropriate construct using Kan/Gen plate. (Done?)(1 day + overnight)<br />
#Liquid culture, miniprep and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (overnight + 1 day)<br />
#Make glycerol stock of RBS-LacZ containing cells. (same day)<br />
<br />
#Ligate the RBS-LacZ construct into double terminator plasmid. (same day)<br />
#Transform ligation and select for construct using Gen/Amp plate. (1 day + overnight)<br />
#Liquid culture, miniprep and confirm using EcoRI/Pst digest (overnight + 1 day)<br />
#prepare glycerol stock (same day)<br />
<br />
<br />
This construct can then be ligated to appropriate promoters for use as a test harness with the substrate 3,4-cyclohexenoesculetin--D-galactopyranoside (S-Gal). For information on conditions see supplementary info from the following paper:<br />
<br />
Nature 438, 441-442 (24 November 2005)<br />
Synthetic biology: Engineering Escherichia coli to see light<br />
Anselm Levskaya1, Aaron A. Chevalier2, Jeffrey J. Tabor2, Zachary Booth Simpson2, Laura A. Lavery2, Matthew Levy2, Eric A. Davidson2, Alexander Scouras2, Andrew D. Ellington2,3, Edward M. Marcotte2,3 and Christopher A. Voigt1,4,5<br />
<br />
<br />
Test harnesses needed:<br />
*OmpR promoter: Ligate to promoter (3 days) and express in Envz+ cells under osmotic stress (time?)<br />
*Red light system: Above construct in Envz- cells with red light system<br />
*Blue light system: Ligate to Promoter responding to ComA (3 days), in Envz- cells constitutively expressing ComA and Blue light receptors (time?)<br />
*AND gate: Use above construct in Envz- cells constitutively expressing Blue light receptor and Red light receptor with ComA expression under the control of OmpR promoter. (time?)</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:GFP_Fluorescent_reporter
Melb:Plan:GFP Fluorescent reporter
2007-08-11T07:41:19Z
<p>Patricia: /* LacZ reporter */</p>
<hr />
<div>==LacZ reporter==<br />
#Ligate LacZ coding region to gentamycin RBS part. (Done?)(1 day)<br />
#Transform and select for appropriate construct using Kan/Gen plate. (Done?)(1 day + overnight)<br />
#Liquid culture, miniprep and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (overnight + 1 day)<br />
#Make glycerol stock of RBS-LacZ containing cells. (same day)<br />
<br />
#Ligate the RBS-LacZ construct into double terminator plasmid. (same day)<br />
#Transform ligation and select for construct using Gen/Amp plate. (1 day + overnight)<br />
#Liquid culture, miniprep and confirm using EcoRI/Pst digest (overnight + 1 day)<br />
#prepare glycerol stock (same day)<br />
<br />
<br />
This construct can then be ligated to appropriate promoters for use as a test harness</div>
Patricia
http://2007.igem.org/wiki/index.php/Melb:Plan:GFP_Fluorescent_reporter
Melb:Plan:GFP Fluorescent reporter
2007-08-11T07:29:17Z
<p>Patricia: </p>
<hr />
<div>==LacZ reporter==<br />
#Ligate LacZ coding region to gentamycin RBS part. (Done?)<br />
#Transform and select for appropriate construct using Kan/Gen plate. (Done?)<br />
#Liquid culture and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (Monday 13/08/07)<br />
#Make glycerol stock of RBS-LacZ containing cells. (Monday 13/08/07)<br />
<br />
#Ligate the RBS-LacZ construct into double terminator plasmid. (Monday 13/08/07)<br />
#Transform ligation and select for construct <br />
#</div>
Patricia