IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/25

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dspB Track

Marianne and Vicki

Transformations

Protocol: See Transformation Protocol in "iGEM common protocols". It is written below for easy access.
Supplies Needed: Competent cells Water bath/heat block at 42°C Ice SOC/LB medium LB agar plates (with appropriate selection)

Steps:

  1. Remove competent cells (100uL aliquots) from -80°C and thaw on ice.
  2. Add 1µL ligation mix to thawed cells and incubate for 30 minutes on ice.
  3. Heat shock in water bath/heat block for 60 seconds and put back on ice for two minutes.
  4. Add 400uL LB medium and incubate at 37C for 2 hours.
  5. Spread plate entire 500µL.
  6. Incubate overnight (<18hrs) at 37°C.
  • Changes:
    • Add 10uL ligation mix
    • Spread on Chloramphenicol plates
  • Used competent cells: DH5α
Table 1. Transformation Mixes = Ligation Mix & competent cells
Transformation SamplesLigation mixCompetent cells DH5α
2T10uL 2L100uL
4T10uL 4L100uL
5T10uL 5L100uL
6T10uL 6L100uL
8T10uL 8L100uL
10T10uL 10L100uL
11T10uL 11L100uL
12T10uL 12L100uL

*Note: Could not add 10uL to 10T because not enough (see previous day)

  • Chose samples 5,6,11,12 because H171 showed distinct bands on the gel verification of the PCR products
  • Chose samples 2,4,8,10 at random - one from each strain: H49 and H80 (of different primers: one with His6-tag and without His6-tag)

Incubate @ 37°C starting 1340
Took out of incubator at 1541

Plated on Chloramphenicol plates - spread 500uL for all

  • Note: Did not fully spread 500uL on 4T (did not have enough for some reason - lacking 0.5uL to 1uL)

Began incubation @ 37°C at 1608
Vicki Ma 04:58, 28 June 2010 (EDT)


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