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.