transfection of plasmids into E. Coli

NEAL MUSTO nmusto01 at gumedlib.dml.georgetown.edu
Fri Mar 3 11:01:38 EST 1995


The simplest protocol I know of is the one we use for such routine
transfection, the freeze/thaw method. We maintain a plate of Coli on LB
plates in the fridge. When we want to stuff in some plasmid, usually a
generous gift from an other investigator, we just loop off about 6
colonies and suspend them in LB broth. The plasmid is added to this,
mixed, incubated on ice for 5-10 min and then frozen in liquid nitrogen
for 1 min. The frozen mix is then thawed on ice (approx 30 min) then .9
ml of warm LB added to bring the volume to 1 ml and the tube incubated
at 37 for 1 hour. We usually get about ten to the 5 colonies per
microgram. Works best when the Coli are fresh, but they can be up to 5
days old and you still get reasonable results.
You can't get much simpler than this.
Neal Musto
Cell Biology
Georgtown University
nmusto01 at gumedlib.dml.georgetown.edu



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