A couple of weeks ago I posted a message saying that I was having a
difficulty preparing E. coli that were acceptably competent (I was getting
about 1x10E6 at most with the Hanahan method). Many of you responded
directly by e-mail and some to this forum, and the consensus was to try the
Inoue et al. method of preparing competent cells (Gene 96, 1990, pp.23-28).
I tried the procedure with my old DH5alpha cells and started the culture at
a rather high density, because they grow SO slowly at 22 degrees (the
protocol says to grow them at 18 degrees) and I still got a 50 fold
improvement in transformation efficiency over the previous method (now at
Next time, I'll get a new bacterial culture and follow the protocol as
closely as I can, and I'm sure the efficiency will improve even more.
Sincerest thanks to everyone for helping me out!
Gulf Coast Res. & Ed. Ctr/ Univ. of FL