Chemical Transformation Protocol

2015-01-13

azim58 - Chemical Transformation Protocol

Chemical Transformation Protocol
(modified from NEB protocol)
http://www.neb.com/nebecomm/products/protocol3.asp

Materials

* competent cells
* SOC
* DNA

Protocol

* Start warming LB plates with appropriate antibiotic (often
  carbinicillin)
* Thaw competent cells on ice (such as DH5-alpha or BL21 for protein
  production)
* Chill DNA or ligation mixture
  o 5 ng (about 2 uL) is probably appropriate

* Add competent cells to the DNA. Mix gently by pipetting up and down
  or flicking the tube 4-5 times to mix the cells and DNA. Do not vortex.
  o original protocol recommends 50uL
  o our lab often uses 10uL when using DH5alpha or STELLAR cells

* place the mixture on ice for 30 minutes. Do not mix
* Heat shock at 42 C for 30 seconds. Do not mix.
* Add room temperature SOC to the tube
  o original protocol recommends 950 uL
  o our lab often uses about 3-5-10X the amount of cells used. Often
    if 10 uL of cells were used 90 uL SOC could be added to bring the
    culture to 100 uL

* Place tube at 37 C for 60 min. Shake vigorously (250 rpm) or rotate.
* Warm selection plates to 37 C
* Spread the cells and ligation mixture onto plates
  o original protocol recommends 50-100 uL
  o our lab often does one plate with 10 uL and one with 90 uL. May
    want to make dilutions in some circumstances e.g. 1 uL to 1000 uL LB
    with 50 uL plated.

* Let the culture soak into the plate for 5-10-20 min.
* Incubate the plates inverted at 37 C overnight.

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