Melbourne/Lab Notebook

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Contents

Week 1

25 June 2007

Prepared LB agar plates.

Transformation

  1. resuspended the following from Registry plates:
    • BBa_I15008 - ho1 (P2 21A, Kan)
    • BBa_I15009 - PcyA (P2 21C, Kan)
    • BBa_R0084 - OmpR positive promoter (P1 11H, Amp)
    1. Punctured foil with pipette tip.
    2. Resuspended in 15uL ddH2O.
    3. Stored in-20 (after taking 1uL for transformation).
  1. Transformed into competent DH5alpha cells with shorter incubation times as follows:
    • 30min on ice after DNA addition
    • 10min on ice after heat shock
    • 30min at 37degrees with LB

26 June 2007

Transformation from Monday

  • Transformation of BBa_I15008 and BBa_I15009 failed. No colonies on plates
  • Small number of colonies on BBa_R0084 plate.
  • Placed in cool room

Transformation

  1. Resuspended the following parts in 15uL:
    1. BBa_B0034 (RBS, plate 1 3O, Amp)
    2. BBa_C0051 (C1 repressor, plate 1 5G, Amp)
    3. BBa_B0010 (Terminator, plate 2 3P, Amp)
  • Think some DNA may have remained in wells
  1. Transformed into competent DH5alpha cells from Joe

Streak plates

  1. Streaked the following cells:
    1. PJS010 (from solid agar, Amp)
    2. Fusion protein (from glycerol stock, Amp?)
    3. BBa_I15010 (from solid agar, Kan)

27 June 2007

Transformation

  1. Repeated transformation of failed parts from Monday:
    1. BBa_I15008 (Kan)
    2. BBa_I15009 (Kan)
  • Used resuspended DNA that was stored on Monday

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:
    1. PJS010
    2. Fusion
    3. BBa_B0034
    4. BBa_C0051
    5. BBa_B0010
    6. BBa_R0084
  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:
    1. PJS010
    2. Fusion
    3. BBa_B0034
    4. BBa_C0051
    5. BBa_B0010
    6. BBa_R0084
    7. BBa_I15010
  2. Stored in -20 freezer

Digest

Liquid culture

  1. Cultured the following cells from transformed plates:
    1. BBa_R0084
    2. BBa_I15010
    3. BBa_B0034
    4. BBa_C0051
    5. BBa_I15008
    6. BBa_I15009

29 June 2007

Miniprep

  1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
    1. BBa_R0084
    2. BBa_I15010
    3. BBa_B0034
    4. BBa_C0051
    5. BBa_I15008
    6. BBa_I15009
  1. Stored in -20 freezer

30 June 2007

Week 2

2 July 2007

Now

3 July 2007

4 July 2007

5 July 2007

6 July 2007

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