Team:Washington/Protocols/Transformation

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==General Transformation Protocol==
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=General Transformation Protocol=
#Thaw 25 - 200 μl TB buffer cells on ice.  Do not use glass tubes, which adsorb DNA.
#Thaw 25 - 200 μl TB buffer cells on ice.  Do not use glass tubes, which adsorb DNA.

Revision as of 22:34, 13 September 2011


General Transformation Protocol

  1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
  2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
  3. Incubate on ice for 30 minutes
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 4 volumes of room temperature SOC (not critical)
  7. Incubate for 1 hour at 37°C on shaker.
    • Note: Can also save some time here by reducing incubation to ~45 min.
    • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
  8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37°C.
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