Melbourne/Lab Notebook Weeks 1-4

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**[[Melbourne/BBa_E0040|'''P1 5H 2''']]
**[[Melbourne/BBa_E0040|'''P1 5H 2''']]
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]
**suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]
 +
*[[Melbourne/4 July 07 Digest|4/7/07 Confirmation of Miniprep Plasmids by digestion]]
<font size=3><b>5 July 2007  
<font size=3><b>5 July 2007  
Line 274: Line 275:
<font color="red"><b>Glycerol Stocks</b></font><BR>
<font color="red"><b>Glycerol Stocks</b></font><BR>
-
The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made and dated 7/7:
+
The following [[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were made and dated 7/7:
*[[Melbourne/BBa_I15010|'''I15010 1''']]  
*[[Melbourne/BBa_I15010|'''I15010 1''']]  
*[[Melbourne/BBa_I15010|'''I15010 2''']]
*[[Melbourne/BBa_I15010|'''I15010 2''']]
Line 311: Line 312:
</b>
</b>
</font><BR>
</font><BR>
-
*Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run
+
*[[Melbourne/Diagnostic Digest|Digested]]  [[Melbourne/BBa_I15010|I15010]] (E/P), [[Melbourne/BBa_R0084|P1 11H]] (E/H), [[Melbourne/BBa_E0241|P2 15L]] (E/P) 1.5hrs run
-
*liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K
+
*[[Melbourne/Growing up cells|liquid culture]] [[Melbourne/BBa_R0084|P1 11H]], [[Melbourne/BBa_I15010|I15010]], [[Melbourne/BBa_R0082|P1 15P]], [[Melbourne/BBa_E0840|P1 16E]], [[Melbourne/BBa_R0083|P1 17H]], [[Melbourne/BBa_Q04510|P2 13K]]
 +
 
 +
*[[Melbourne/Loading a DNA gel|Loaded and ran Gel]]:
 +
*# [[Melbourne/primary DNA marker|DNA ladder 1kb+]]
 +
*# E/P [[Melbourne/BBa_I15010|I15010]]
 +
*# E/P [[Melbourne/BBa_I15010|I15010]]
 +
*# E/H [[Melbourne/BBa_R0084|P1 11H]]
 +
*# E/H [[Melbourne/BBa_R0084|P1 11H]]
 +
*# Undigested [[Melbourne/BBa_E0241|P2 15L]]
 +
*# Undigested [[Melbourne/BBa_E0241|P2 15L]]
 +
*# E [[Melbourne/BBa_E0241|P2 15L]]
 +
*# E [[Melbourne/BBa_E0241|P2 15L]]
 +
*# P [[Melbourne/BBa_E0241|P2 15L]]
 +
*# P [[Melbourne/BBa_E0241|P2 15L]]
 +
*# E/P [[Melbourne/BBa_E0241|P2 15L]]
 +
*# E/P [[Melbourne/BBa_E0241|P2 15L]]
 +
*# [[Melbourne/primary DNA marker|DNA ladder 1kb+]]
 +
 
 +
Ran for 1.5 hours
 +
 
<font size=3><b>10 July 2007  
<font size=3><b>10 July 2007  
</b>
</b>
</font><BR>
</font><BR>
-
*Miniprep
+
*[[Melbourne/Miniprep protocol|Miniprep]]
-
*Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H  1.0hrs run
+
*[[Melbourne/Diagnostic Digest|Digested]] [[Melbourne/BBa_E0840|P1 16E]], [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_Q04510|P2 13K]], [[Melbourne/BBa_I15010|I15010]], [[Melbourne/BBa_R0082|P1 15P]], [[Melbourne/BBa_R0084|P1 11H]], [[Melbourne/BBa_R0083|P1 17H]]  
-
*liquid culture I15010, P2
+
*[[Melbourne/Growing up cells|liquid culture]] [[Melbourne/BBa_I15010|I15010]], [[Melbourne/BBa_Q04510|P2 13K]]
 +
 
 +
*[[Melbourne/Loading a DNA gel|Loaded and ran Gel]]:
 +
*# [[Melbourne/primary DNA marker|DNA ladder 1kb+]]
 +
*# E/P [[Melbourne/BBa_E0840|P1 16E]]
 +
*# E/P [[Melbourne/BBa_E0840|P1 16E]]
 +
*# E/P [[Melbourne/BBa_E0430|P1 11A]]
 +
*# E/P [[Melbourne/BBa_E0430|P1 11A]]
 +
*# E/P [[Melbourne/BBa_Q04510|P2 13K]]
 +
*# E/P [[Melbourne/BBa_Q04510|P2 13K]]
 +
*# E/P [[Melbourne/BBa_I15010|I15010]]
 +
*# E/H [[Melbourne/BBa_R0082|P1 15P]]
 +
*# E/H [[Melbourne/BBa_R0082|P1 15P]]
 +
*# E/H [[Melbourne/BBa_R0084|P1 11H]]
 +
*# E/H [[Melbourne/BBa_R0084|P1 11H]]
 +
*# E/H  [[Melbourne/BBa_R0083|P1 17H]]
 +
*# E/H  [[Melbourne/BBa_R0083|P1 17H]]
 +
*#[[Melbourne/primary DNA marker|DNA ladder 1kb+]]
 +
 
 +
Ran at 95V for 1 hour
 +
 
<font size=3><b>11 July 2007  
<font size=3><b>11 July 2007  
</b>
</b>
</font><BR>
</font><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
+
*[[Melbourne/Diagnostic Digest|Digested]] for ligation [[Melbourne/BBa_R0082|P1 15P]](1)10/7, [[Melbourne/BBa_R0084|P1 11H]] 10/7, [[Melbourne/BBa_R0083|P1 17H]](1)10/7, [[Melbourne/BBa_E0840|P1 16E]](2)10/7, [[Melbourne/BBa_E0430|P1 11A]](1)10/7
-
*loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H
+
*[[Melbourne/Loading a DNA gel|Loaded]]:
-
** Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.
+
*# X/P [[Melbourne/BBa_E0430|P1 11A]]
-
** Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA
+
*#X/P [[Melbourne/BBa_E0840|P1 16E]]
-
*Excise bands of interest and purify invitrogen
+
*#S/P [[Melbourne/BBa_R0082|P1 15P]]
-
*liquid culture P1-11A,P1-15P 10ml
+
*#S/P [[Melbourne/BBa_R0084|P1 11H]]
-
*Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.
+
 
-
*Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H
+
*Excise bands of interest and purify using invitrogen kit
 +
*[[Melbourne/Growing up cells|liquid culture]] [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_R0082|P1 15P]] 10ml
 +
*[[Melbourne/Transformation Protocol|Transformed]] [[Melbourne/BBa_P1010_AC|P2 21B]], [[Melbourne/BBa_P1010_AK|P2 23N]], [[Melbourne/BBa_P1010_A|P3 20I]] into DB3.1 heat shock.
 +
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_E0840|P1 16E]], [[Melbourne/BBa_R0084|P1 11H]], [[Melbourne/BBa_R0082|P1 15P]], [[Melbourne/BBa_R0083|P1 17H]]
 +
 
 +
 
<font size=3><b>12 July 2007  
<font size=3><b>12 July 2007  
</b>
</b>
</font><BR>
</font><BR>
-
*Ran Gel P1-11A,P1-16E,P1-15P,P1-11H
+
*[[Melbourne/Loading a DNA gel|Ran Gel]]:
-
*miniprepped P1-11A,P1-15P
+
*# 20kB+ ladder
-
*Digest
+
*#[[Melbourne/BBa_E0430|P1 11A]]
-
*Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)
+
*#[[Melbourne/BBa_E0840|P1 16E]]
-
*Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)
+
*#[[Melbourne/BBa_R0082|P1 15P]]
-
*Liquid culture transformants 11/7
+
*#[[Melbourne/BBa_R0084|P1 11H]]
 +
(samples from gel purified DNA of 11/7)
 +
 
 +
*[[Melbourne/Miniprep protocol|Minipreped]] [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_R0082|P1 15P]]
 +
*[[Melbourne/Diagnostic Digest|Digest]]:
 +
*# S/P+ Alkaline Phosphatase of [[Melbourne/BBa_R0082|P1 15P]]
 +
*# X/P of [[Melbourne/BBa_E0430|P1 11A]]
 +
 
 +
-Did not work: too much DNA.
 +
 
 +
*[[Melbourne/Ligation Protocol|Ligate]] control=(2uL ligase buffer,1uL ligase,5uL vector([[Melbourne/BBa_R0082|P1 15P]]),12uL H20)
 +
*[[Melbourne/Ligation Protocol|Ligate]] (2uL ligase buffer,1uL ligase,5uL vector([[Melbourne/BBa_R0082|P1 15P]]),10uL insert([[Melbourne/BBa_E0430|P1 11A]]),2uL H20)
 +
*[[Melbourne/Growing up cells|liquid culture]] transformants 11/7
 +
 
 +
 
<font size=3><b>13 July 2007  
<font size=3><b>13 July 2007  
</b>
</b>
</font><BR>
</font><BR>
-
*miniprep cultures from transformants 11/7
+
*[[Melbourne/Miniprep protocol|Minipreped]] cultures from transformants 11/7
-
*Digestion P1-15P and P1-11A from 12/7/07  37degC 3hours 15 minutes stopped 5uL of 6X loading dye.
+
*[[Melbourne/Diagnostic Digest|Digested]]
-
*Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.
+
*# S/P [[Melbourne/BBa_R0082|P1 15P]]
-
*Glycerol stocks of P3-20I,P2-21B,P2-23N
+
*# X/P [[Melbourne/BBa_E0430|P1 11A]]
-
*Transform DH5a with ligation product
+
from 12/7/07   
 +
37degC 3hours 15 minutes stopped by 5uL of 6X [[Melbourne/primary dna loading|Loading dye]].
 +
 
 +
*[[Melbourne/Loading a DNA gel|Ran on Gel]] for 1 hour
 +
*Excise bands 800bp from [[Melbourne/BBa_E0430|P1 11A]], 2Kbp from [[Melbourne/BBa_R0082|P1 15P]] and purified.
 +
*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] of [[Melbourne/BBa_P1010_A|P3 20I]], [[Melbourne/BBa_P1010_AC|P2 21B]], [[Melbourne/BBa_P1010_AK|P2 23N]]
 +
*[[Melbourne/Transformation Protocol|Transform]] DH5a with ligation product from 12/7.
 +
 
 +
 
<font size=3><b>14 July 2007  
<font size=3><b>14 July 2007  
</b>
</b>
</font><BR>
</font><BR>
-
*Transform using 10uL of ligation reaction
+
*[[Melbourne/Transformation Protocol|Transformation]]
 +
 
 +
-results:
 +
6 colonies on control plate<BR>
 +
5 colonies on ligation plate<BR>
 +
6 colonies on transformation of digested DNA from Thursday
==Week 4==
==Week 4==
Line 356: Line 428:
</b>
</b>
</font><BR>
</font><BR>
-
*[[Melbourne/colony pcr|Colony PCR]]
+
*[[Melbourne/Colony PCRl|Colony PCR]] of all (5) colonies from ligation plate (14/7) and one from control plate.
 +
*[[Melbourne/Diagnostic Digest|Digested]] same colonies at the same time to confirm that colony PCR was working.
 +
<BR> -results: No colonies were apparent.
 +
 
 +
<font size=3><b>17 July 2007
 +
</b>
 +
</font><BR>
 +
 
 +
*Set up repeat of [[Melbourne/Ligation Protocol|Ligation]] from 12/7.
 +
 
<font size=3><b>18 July 2007  
<font size=3><b>18 July 2007  
</b>
</b>
</font><BR>
</font><BR>
-
*Purchased XbaI,EcoRI,PstI
+
*Purchased [[Melbourne/primary Restriction enzymes|Restriction enzymes]] XbaI, EcoRI, PstI
-
*Miniprepped cultures 1,3,6 from 16/7
+
*[[Melbourne/Miniprep protocol|Miniprepped]] cultures 1,3,6 from 16/7
-
*Digestion with E/P
+
*[[Melbourne/Diagnostic Digest|Digested]] with E/P in triplicate.
-
*Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?
+
*[[Melbourne/Loading a DNA gel|Ran Gel]]:
 +
*# [[Melbourne/primary DNA marker|DNA ladder]],
 +
*# ctrl (from PCR reaction 1 earlier)
 +
*# Digest 1 ([[Melbourne/BBa_R0082|P1 15P]])
 +
*# Digest 3 ([[Melbourne/BBa_R0082|P1 15P]])
 +
*# Digest 6 ([[Melbourne/BBa_R0082|P1 15P]])
 +
*# Digest from 12/7 1([[Melbourne/BBa_E0430|P1 11A]])
 +
*# Digest from 12/7 2([[Melbourne/BBa_E0430|P1 11A]])
 +
 
 +
*[[Melbourne/Transformation Protocol|Transformed]]
 +
*#[[Melbourne/Lab Notebook gv 1|pN26]]
 +
*#[[Melbourne/Lab Notebook gv 1|pN29]]
 +
with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways.
 +
 
 +
-observe B+P plates clean. Small colonies on NL29 and NL26 plates as well as C.
 +
 
 +
*Picked three colonies from each plate and [[Melbourne/Growing up cells|cultured.]]
 +
 
 +
<font size=3><b>20 July 2007
 +
</b>
 +
</font><BR>
 +
 
 +
*[[Melbourne/Transformation Protocol|Transformation]] of ligations from 17/7 together with a control of undigested [[Melbourne/BBa_R0082|P1 15P]] and competent cells only.
 +
 
 +
 
 +
<font size=3><b>21 July 2007
 +
</b>
 +
</font><BR>
 +
 
 +
*Transformation results from 20/7: 0 colonies on competent cells only control, many on undigested vector, and 3 on ligation plate.

Latest revision as of 03:43, 25 October 2007

<Return to Lab notebook> <team home page>

Contents

Week 1


  • 26 June 2007: Streaked the following cells:
    1. pJS010 (from solid agar, Amp)
    2. Fusion protein (from glycerol stock, Amp?)
    3. BBa_I15010 (from solid agar, Kan)


Liquid culture

  1. Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
  2. Aliquoted 5mL Amp LB into 6 50mL falcon tubes
  3. To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
  4. To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
  5. Cells incubated at 37degrees with shaking overnight.

28 June 2007

Miniprep

  1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
  2. Stored in -20 freezer


Liquid culture

  1. Cultured the following cells from transformed plates:

29 June 2007
Miniprep

  1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
  1. Stored in -20 freezer

Week 2

  • 3 July 2007: Re Transformed into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
    1. P4 8J -> Three colonies -> grew in liquid culture 4 july
    2. P1 5H -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'
    3. P2 15L -> Three colonies -> grew in liquid culture 4 july
  • 4 July 2007:

Ampicillin Plates

  • Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June.
    • Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.

Transformation
Transformed the following and grew on new ampicillin plates

  • P1 5H
  • P4 8J
  • P2 15L
    • Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)

Liquid Culture

5 July 2007

Miniprep


Digest
Performed the following digests on DNA from the above miniprep

EcoR1/Pst1 with buffer 3

EcoR1/HaeII in buffer 2

XbaI/SpeI in buffer 2

Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20

Transformation

Liquid Culture

6 July 2007

Digest Gel

Miniprep

  • Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7
  • Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.
  • P1 5H 1
  • P1 5H 2
  • P4 8J 1
  • P4 8J 2
  • P2 15L 1
  • P2 15L 2


Digest

  • Digested 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.
  • Incubated for 2hours 25min at 37degrees
  • Added 5uL 6x loading dye and stored at -20

Glycerol Stocks
The following Glycerol Stocks were made:

Stored at -80

Liquid Culture
Cultured the following in 5mL LB

Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7

7 July 2007

Digest Gel

Glycerol Stocks
The following Glycerol Stocks were made and dated 7/7:

Stored at -80

Miniprep

  • Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.

Digest

8 July 2007

Transformation
Resuspended and transformed the following

  • P1 15P(BBa_R0082, Omp R+, Amp)
  • P1 17H(BBa_R0083, truncated BBa_R0082 Omp R+, Amp)
  • P1 11A(BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
  • P1 16E(BBa_E0430; RBS,GFP,term; Amp)

The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.

  • P2 13K (BBa_Q04510, c1 inverter, Kan)

Week 3

9 July 2007

Ran for 1.5 hours

10 July 2007

Ran at 95V for 1 hour

11 July 2007


12 July 2007

(samples from gel purified DNA of 11/7)

-Did not work: too much DNA.


13 July 2007

from 12/7/07 37degC 3hours 15 minutes stopped by 5uL of 6X Loading dye.


14 July 2007

-results: 6 colonies on control plate
5 colonies on ligation plate
6 colonies on transformation of digested DNA from Thursday

Week 4

16 July 2007

  • Colony PCR of all (5) colonies from ligation plate (14/7) and one from control plate.
  • Digested same colonies at the same time to confirm that colony PCR was working.


-results: No colonies were apparent.

17 July 2007

18 July 2007

with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways.

-observe B+P plates clean. Small colonies on NL29 and NL26 plates as well as C.

  • Picked three colonies from each plate and cultured.

20 July 2007

  • Transformation of ligations from 17/7 together with a control of undigested P1 15P and competent cells only.


21 July 2007

  • Transformation results from 20/7: 0 colonies on competent cells only control, many on undigested vector, and 3 on ligation plate.