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''']]
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*[[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  
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*[[Melbourne/Transformation Protocol|Transformed]]  
*[[Melbourne/Transformation Protocol|Transformed]]  
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*#[[Melbourne/N26|NL26]]  
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*#[[Melbourne/Lab Notebook gv 1|pN26]]
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*#[[Melbourne/N29|NL29]]
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*#[[Melbourne/Lab Notebook gv 1|pN29]]
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  with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways.  
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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.
-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.]]
*Picked three colonies from each plate and [[Melbourne/Growing up cells|cultured.]]
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<font size=3><b>20 July 2007
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*[[Melbourne/Transformation Protocol|Transformation]] of ligations from 17/7 together with a control of undigested [[Melbourne/BBa_R0082|P1 15P]] and competent cells only.
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<font size=3><b>21 July 2007
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*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.
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