Melbourne/Lab Notebook

From 2007.igem.org

(Difference between revisions)
(Week 2)
(Week 2)
Line 253: Line 253:
===7 July 2007===
===7 July 2007===
 +
 +
*Made 10x TAE buffer
====Digest Gel====
====Digest Gel====
Line 284: Line 286:
*Incubated for 3hours at 37degrees
*Incubated for 3hours at 37degrees
*Added 5uL 6x loading dye and stored at -20
*Added 5uL 6x loading dye and stored at -20
-
 
===8 July 2007===
===8 July 2007===
 +
 +
====Transformation====
 +
====Tranformation====
 +
Resuspended and [[Melbourne/Transformation Protocol|transformed]] the following
 +
*[[Melbourne/BBa_R0082|'''P1 15P''']](BBa_R0082, Omp R+, Amp)
 +
*[[Melbourne/BBa_R0083|'''P1 17H''']](BBa_R0083, truncated BBa_R0082 Omp R+, Amp)
 +
*[[Melbourne/BBa_E0430|'''P1 11A''']](BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
 +
*[[Melbourne/BBa_E0840|'''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.
 +
*[[Melbourne/BBa_Q04510|'''P2 13K''']] (BBa_Q04510, c1 inverter, Kan)
==Week 3==
==Week 3==

Revision as of 23:35, 8 July 2007

<Back to 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

Digest

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

30 June 2007

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.

Tranformation

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

EcoRI/PstI with buffer 3
EcoRI/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

  • Made 10x TAE buffer

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

Tranformation

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

10 July 2007

11 July 2007

12 July 2007

13 July 2007

Week 4

16 July 2007

17 July 2007

18 July 2007

19 July 2007

20 July 2007

Week 5

23 July 2007

24 July 2007

25 July 2007

26 July 2007

27 July 2007

Week 6

30 July 2007

31 July 2007

1 Aug 2007

2 Aug 2007

3 Aug 2007