Melbourne/Lab Notebook Weeks 1-4

From 2007.igem.org

(Difference between revisions)
(Week 3)
(Week 3)
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*[[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
*[[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
*[[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/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]]
-
<br>
+
 
*[[Melbourne/Loading a DNA gel|Loaded and ran Gel]]:  
*[[Melbourne/Loading a DNA gel|Loaded and ran Gel]]:  
<br>1. [[Melbourne/primary DNA marker|DNA ladder 1kb+]]<br>2. E/P [[Melbourne/BBa_I15010|I15010]] <br> 3. E/P [[Melbourne/BBa_I15010|I15010]] <br>4. E/H [[Melbourne/BBa_R0084|P1 11H]]<br> 5. E/H [[Melbourne/BBa_R0084|P1 11H]]<BR> 6. Undigested [[Melbourne/BBa_E0241|P2 15L]] <BR> 7. Undigested [[Melbourne/BBa_E0241|P2 15L]] <BR> 8. E [[Melbourne/BBa_E0241|P2 15L]] <BR> 9. [[Melbourne/BBa_E0241|P2 15L]] <BR> 10. P [[Melbourne/BBa_E0241|P2 15L]] <BR> 11. P [[Melbourne/BBa_E0241|P2 15L]] <BR> 12. E/P [[Melbourne/BBa_E0241|P2 15L]] <BR> 13. E/P [[Melbourne/BBa_E0241|P2 15L]] <BR> 14. [[Melbourne/primary DNA marker|DNA ladder 1kb+]]
<br>1. [[Melbourne/primary DNA marker|DNA ladder 1kb+]]<br>2. E/P [[Melbourne/BBa_I15010|I15010]] <br> 3. E/P [[Melbourne/BBa_I15010|I15010]] <br>4. E/H [[Melbourne/BBa_R0084|P1 11H]]<br> 5. E/H [[Melbourne/BBa_R0084|P1 11H]]<BR> 6. Undigested [[Melbourne/BBa_E0241|P2 15L]] <BR> 7. Undigested [[Melbourne/BBa_E0241|P2 15L]] <BR> 8. E [[Melbourne/BBa_E0241|P2 15L]] <BR> 9. [[Melbourne/BBa_E0241|P2 15L]] <BR> 10. P [[Melbourne/BBa_E0241|P2 15L]] <BR> 11. P [[Melbourne/BBa_E0241|P2 15L]] <BR> 12. E/P [[Melbourne/BBa_E0241|P2 15L]] <BR> 13. E/P [[Melbourne/BBa_E0241|P2 15L]] <BR> 14. [[Melbourne/primary DNA marker|DNA ladder 1kb+]]
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*[[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
*[[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
-
*[[Melbourne/Loading a DNA gel|Loaded]] order X/P [[Melbourne/BBa_E0430|P1 11A]], X/P [[Melbourne/BBa_E0840|P1 16E]],S/P [[Melbourne/BBa_R0082|P1 15P]],S/P [[Melbourne/BBa_R0084|P1 11H]]
+
*[[Melbourne/Loading a DNA gel|Loaded]]:
 +
*# X/P [[Melbourne/BBa_E0430|P1 11A]]
 +
*#X/P [[Melbourne/BBa_E0840|P1 16E]]
 +
*#S/P [[Melbourne/BBa_R0082|P1 15P]]
 +
*#S/P [[Melbourne/BBa_R0084|P1 11H]]
 +
 
*Excise bands of interest and purify using invitrogen kit
*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/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/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]]
*[[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  
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-
*[[Melbourne/Loading a DNA gel|Ran Gel]] [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_E0840|P1 16E]], [[Melbourne/BBa_R0082|P1 15P]], [[Melbourne/BBa_R0084|P1 11H]]
+
*[[Melbourne/Loading a DNA gel|Ran Gel]]:
 +
*# 20kB+ ladder
 +
*#[[Melbourne/BBa_E0430|P1 11A]]
 +
*#[[Melbourne/BBa_E0840|P1 16E]]
 +
*#[[Melbourne/BBa_R0082|P1 15P]]
 +
*#[[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/Miniprep protocol|Minipreped]] [[Melbourne/BBa_E0430|P1 11A]], [[Melbourne/BBa_R0082|P1 15P]]
-
*[[Melbourne/Diagnostic Digest|Digest]]
+
*[[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]] 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/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
*[[Melbourne/Growing up cells|liquid culture]] transformants 11/7
 +
 +
<font size=3><b>13 July 2007  
<font size=3><b>13 July 2007  
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*[[Melbourne/Miniprep protocol|Minipreped]] cultures from transformants 11/7
*[[Melbourne/Miniprep protocol|Minipreped]] cultures from transformants 11/7
-
*[[Melbourne/Diagnostic Digest|Digested]] [[Melbourne/BBa_R0082|P1 15P]] and [[Melbourne/BBa_E0430|P1 11A]] from 12/7/07  37degC 3hours 15 minutes stopped 5uL of 6X [[Melbourne/primary dna loading|Loading dye]].
+
*[[Melbourne/Diagnostic Digest|Digested]]  
 +
*# S/P [[Melbourne/BBa_R0082|P1 15P]]
 +
*# X/P [[Melbourne/BBa_E0430|P1 11A]]  
 +
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.
*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/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
+
*[[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  
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Revision as of 11:19, 8 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


1. DNA ladder 1kb+
2. E/P I15010
3. E/P I15010
4. E/H P1 11H
5. E/H P1 11H
6. Undigested P2 15L
7. Undigested P2 15L
8. E P2 15L
9. P2 15L
10. P P2 15L
11. P P2 15L
12. E/P P2 15L
13. E/P P2 15L
14. DNA ladder 1kb+

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

  • Transform using 10uL of ligation reaction

Week 4

16 July 2007

18 July 2007

  • Purchased XbaI,EcoRI,PstI
  • Miniprepped cultures 1,3,6 from 16/7
  • Digestion with E/P
  • Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?