Melbourne/Lab Notebook Weeks 1-4

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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

10 July 2007

11 July 2007

  • 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
  • loaded order X/P P1 11A, X/P P1-16E,S/P P1-15P,S/P P1 11H
    • Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.
    • Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA
  • Excise bands of interest and purify invitrogen
  • liquid culture P1-11A,P1-15P 10ml
  • Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.
  • Glycerol stocks P1 11A, P1-16E, P1 11H, P1-15P,P1-17H

12 July 2007

  • Ran Gel P1-11A ,P1-16E, P1-15P, P1 11H
  • miniprepped P1 11A, P1-15P
  • Digest
  • Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)
  • Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1 11A),2uL H20)
  • Liquid culture transformants 11/7

13 July 2007

  • miniprep 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.
  • Excise bands 800bp from P1 11A, 2Kbp from P1-15P and purified.
  • Glycerol stocks of P3-20I,P2-21B,P2-23N
  • Transform DH5a with ligation product

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?
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