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
 * 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.
 * original protocol recommends 50uL
 * 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
 * original protocol recommends 950 uL
 * 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
 * original protocol recommends 50-100 uL
 * 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.