IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation protocol 1

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Transformation Protocol 1

Equipment

Reagents

SpII:

  • 200ml T base,
  • 50% (w/v) glucose 2ml,
  • 1.2% (w/v) MgSO4.7H2O,
  • 1% (w/v) casamino acids 2ml,
  • 10% (w/v) bacto yeast extract 2ml,
  • 0.1M CaCl2 1ml,
  • Antibiotic growth selectors.

T base per litre:

  • 2g (NH4)2SO4,
  • 18.3g K2HPO4.3H2O,
  • 6g KH2PO4,
  • 6g trisodium citrate 2H2O

SpC:

  • 20ml T base,
  • 50%(w/v) glucose 0.2ml,
  • 1.2% (w/v) MgSO4.7H2O 0.3ml,
  • 10% (w/v) Bacto yeast extract 0.4ml,
  • 1% (w/v) casamino acids 0.5ml,
  • growth selectors

SpII + EGTA:

  • 200ml SpII,
  • 4ml EGTA (0.1M, pH 8.0,0.152g),
  • Store in frozen small aliquots(20ml),

Protocol

Day 1

  • Inoculate 20ml of LB media with a single colony from an LB agar plate of B.subtilis and grow overnight at 30oC.

Day 2

  • Remove 1ml of the overnight solution and place in a curvette, then quickly measure the OD600 of the solution. Using the following calculation work out the dilution required to get an OD600 of 0.5 in 20ml.
  • Add the required volume of overnight solution to 20ml of pre-warmed SpC medium. Incubate this culture at 37ºC with vigorous aeration and take periodic OD readings (OD600) to assess cell growth. When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 minutes) inoculate 200ml of pre-warmed SpII medium with 2ml of stationary-phase culture and continue incubation at 37ºC with slower aeration.
  • After 90 minutes incubation, pellet the cells by centrifugation (8000g, 5 minutes) at room temperature.
  • After centrifugation remove the supernatant and save, then gently resuspend the cell pellet in 18ml of the saved supernatant and add 2ml of sterile glycerol; mix gently.
  • Aliquot the competent cells (0.5ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-ice/ethanol bath and store at -70ºC.

Day 3

  • Thaw competent cells rapidly by immersing frozen tubes in a 37ºC water bath,
  • Immediately, add one volume (20ml I think) of SpII + EGTA to the thawed cells 0.5ml,
  • In a sterile test tube add competent cells (0.2-0.5ml) to the DNA solution (<0.1ml) and incubate in a roller drum at 37ºC.
  • Dilute the transformed cells as appropriate in T Base containing 0.5% glucose and plate immediately onto selective media.


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