IGEM:PennState/2006/Transformation

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< IGEM:PennState | 2006
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Contents

Transformation

Time: 2.25 hr

Media/Reagents

  • Competent cells
  • SOC1
  • Antibiotic LB plates

Pre

  • Deactivate the ligase by heating at 65 degrees for 20 minutes.
  • Chill ligated DNA on ice
  • Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.

Transformation

  1. Add 3 μL ligation mixture to cells in bottom of tube
  2. Incubate on ice for 30 min
  3. Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
  4. Incubate on ice for 2 min
  5. Add 500 μL warm SOC media
  6. Shake in 37ºC room for 1 hr
  7. Spread 200 μL on LB/antibiotic plates using rotator and glass spreading wand.
  8. Incubate plates in 37ºC room overnight.


1To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)

Cloning

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