Glasgow/Wetlab/Week3

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!align=center|[[Image:Uog.jpg]] ||  [[Glasgow|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Main Page</font>]] || [[Glasgow/Wetlab|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Wetlab Log</font>]] ||  [[Glasgow/Modeling|<font face=georgia color=#3366CC size=4>Go To <br> Glasgow's <br>Drylab Log</font>]]
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----
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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|- align=center
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|[https://2007.igem.org/Glasgow/Wetlab/Protocols <font face=georgia color=#3366CC size=5><b>Protocols</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/References <font face=georgia color=#3366CC size=5><b>References</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Resources <font face=georgia color=#3366CC size=5><b>Resources</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Orders <font face=georgia color=#3366CC size=5><b>Orders</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Biobricks <font face=georgia color=#3366CC size=5><b>Biobricks<br>Used</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Gels <font face=georgia color=#3366CC size=5><b>Gels</b></font>]
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|}
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----
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== Week 3 ==
== Week 3 ==
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=== Tuesday 17/07 ===
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=== Tuesday 17th July 2007 ===
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#Restriction Digests (see [[Glasgow/Wetlab/Protocols#Protocol 7: Restriction Digests|Protocol 7]]):
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#Restriction Digests of transformed BioBricks (see [[Glasgow/Wetlab/Protocols#Protocol 7: Restriction Digests|Protocol 7]]):
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#*BBa_J23119 (strong constitutive promoter) pSB1A2.  1 x NheI, 1 x PvuI.  680bp, 1430bp
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{| border="1" cellspacing="0" cellpadding="5" align="center"
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#*BBa_R0062 (HSL and luxR) pBB1A2.  1 x EcoRI, 1 x PvuI. 1460bp, 660bp
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!Label
-
#*BBa_J04500 (IPTG inducer and RBS) pSB1AK3.  1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
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!BioBrick
-
#*BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.
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!Plasmid
-
#*BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
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!Description
-
#*BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
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!Enzymes
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!Expected Sizes
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|-
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|3/19A
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|BBa_J23119  
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|pSB1A2
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|strong constitutive promoter
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|NheI, PvuI  
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|680bp, 1430bp
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|-
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|1/9G
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|BBa_R0062  
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|pBB1A2
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|HSL and luxR
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|EcoRI, PvuI
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|1460bp, 660bp
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|-
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|1/16P
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|BBa_J04500
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|pSB1AK3
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|IPTG inducer and RBS
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|PvuII, PvuI
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|2250bp, 1030bp, 730bp
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|-
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|4/11C
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|BBa_p1010
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|pSB3K3
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|death gene
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|BamHI, XhoI
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|190bp, 2390bp,840bp
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|-
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|1/5H
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|BBa_E0040  
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|pSB1A2
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|GFP no promoter
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|Hime II, PvuI  
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|1630bp, 1170bp
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|-
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|4/6D
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|BBa_I52001
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| p5B4K5
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|High copy death gene
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|AvaI, PvuI
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|920bp, 1470bp, 2120bp
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|}
-
=== Wednesday 18/07 ===
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=== Wednesday 18th July 2007 ===
#Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
#Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
-
#Maija ran a gel of the restriction digests to check the sizes of the biobricks.  These were the maps we made yesterday.
+
#[[User:MaijaP|Maija]] ran a gel of the restriction digests to check the sizes of the biobricks.  These were the maps we made yesterday.
-
#Maia is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see [[Glasgow/Wetlab/Protocols#Protocol 8: MoBio PowersoilTM DNA Purification Kit|Protocol 8]]); only change was to shorten "shaking" time from 10 to 2 minutes.
+
#[[User:Mojs|Maia]] is extracting DNA from 3 samples of Pseudomonas Aeruginosa, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see [[Glasgow/Wetlab/Protocols#Protocol 8: MoBio PowersoilTM DNA Purification Kit|Protocol 8]]); only change was to shorten "shaking" time from 10 to 2 minutes.
-
#PCR trial run using Reddymix and Touch 2 done on:
+
#PCR trial run using Reddymix and Touch 2 done (see [[Glasgow/Wetlab/Protocols#Protocol 9: PCR|Protocol 9]] for PCR).
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#*pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
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{| border="1" cellspacing="0" cellpadding="5" align="center"
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#*pQF52 plasmid: DntR_prefix / DntR_suffix
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!Template
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#*DmpR WT/24: DntR_prefix / DntR_suffix
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!Primers
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#*Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
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|-
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#*See [[Glasgow/Wetlab/Protocols#Protocol 9: PCR|Protocol 9]] for PCR.
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|pGLTUR  
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|
 +
<br>XylR_prefix + XylR_suffix
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<br>Pr_prefix + Pr_suffix  
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<br>Pr_prefix + XylR_suffix
 +
<br>Pu_prefix_EM + Pu_suffix_EM
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|-
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|pQF52  
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|DntR_prefix + DntR_suffix
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|-
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|DmpR WT/24  
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|DntR_prefix + DntR_suffix
 +
|}
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#*Biobricks: [http://partsregistry.org/Part:BBa_R0062 R0062], [http://partsregistry.org/Part:BBa_E0040 E0040], [http://partsregistry.org/Part:BBa_J04500 J04500], [http://partsregistry.org/Part:BBa_J23119 J23119], [http://partsregistry.org/Part:BBa_p1010 p1010], [http://partsregistry.org/Part:BBa_I52001 I52001].
-
=== Thursday 19/07 ===
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=== Thursday 19th July 2007 ===
-
#Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See [[Glasgow/Wetlab/Protocols#Protocol 9: PCR|Protocol 9]]) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
+
#[[User:Mojs|Maia]] redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (see [[Glasgow/Wetlab/Protocols#Protocol 9: PCR|Protocol 9]]) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
#*XylR prefix and XylR suffix
#*XylR prefix and XylR suffix
#*Pr prefix and Pr suffix
#*Pr prefix and Pr suffix
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#*DntR prefix and DntR suffix 2
#*DntR prefix and DntR suffix 2
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=== Friday 20/07 ===
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=== Friday 20th July 2007 ===
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<br>
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{| valign=top cellpadding=3
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|-
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![[Glasgow/Wetlab/Week2|<font face=georgia color=#3366CC size=4>Previous  <br>  Week</font>]] || [[Glasgow/Wetlab/Week4|<font face=georgia color=#3366CC size=4>Next <br>  Week</font>]]
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|-
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|}
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----
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{| valign=top cellpadding=3
 +
|-
 +
!align=center|[[Image:Uog.jpg]] ||  [[Glasgow|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Main Page</font>]] || [[Glasgow/Wetlab|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Wetlab Log</font>]]
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|}

Latest revision as of 17:08, 22 October 2007

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Used 		Gels

Contents

Week 3

Tuesday 17th July 2007

  1. Restriction Digests of transformed BioBricks (see Protocol 7):
Label BioBrick Plasmid Description Enzymes Expected Sizes
3/19A BBa_J23119 pSB1A2 strong constitutive promoter NheI, PvuI 680bp, 1430bp
1/9G BBa_R0062 pBB1A2 HSL and luxR EcoRI, PvuI 1460bp, 660bp
1/16P BBa_J04500 pSB1AK3 IPTG inducer and RBS PvuII, PvuI 2250bp, 1030bp, 730bp
4/11C BBa_p1010 pSB3K3 death gene BamHI, XhoI 190bp, 2390bp,840bp
1/5H BBa_E0040 pSB1A2 GFP no promoter Hime II, PvuI 1630bp, 1170bp
4/6D BBa_I52001 p5B4K5 High copy death gene AvaI, PvuI 920bp, 1470bp, 2120bp

Wednesday 18th July 2007

  1. Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
  2. Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
  3. Maia is extracting DNA from 3 samples of Pseudomonas Aeruginosa, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Protocol 8); only change was to shorten "shaking" time from 10 to 2 minutes.
  4. PCR trial run using Reddymix and Touch 2 done (see Protocol 9 for PCR).
Template Primers
pGLTUR


XylR_prefix + XylR_suffix
Pr_prefix + Pr_suffix
Pr_prefix + XylR_suffix
Pu_prefix_EM + Pu_suffix_EM

pQF52 DntR_prefix + DntR_suffix
DmpR WT/24 DntR_prefix + DntR_suffix

Thursday 19th July 2007

  1. Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (see Protocol 9) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
    • XylR prefix and XylR suffix
    • Pr prefix and Pr suffix
    • Pr prefix and XylR suffix
    • Pu prefix and Pu suffix
    • DntR prefix and DntR suffix 2

Friday 20th July 2007


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