I've posted this problem before but have a bit of new information to add
to the problem. I am using Kunkel selection site-directed mutagenesis
from an M13 derived template. We have used this method for years in the
lab with success. However, during the past year, I have not been able to
get plaques after transfecting with the extension reaction. Controls show
that my cells are competent and contain the F-factor. Direct
incorporation of P32-dATP in the extension shows that I am getting
extension. However, I always see two bands of equal intensity in the
autoradiogram of the extension reaction.
My first thought was that the two bands were the result of partial
linearization of the ssDNA template. Several template preps show the same
pattern, however, so I'm not sure.
Any ideas? It seems even if I'm getting two bands I should still get some
plaques from the transfection. Any help would be greatly appreciated.
Texas A&M University
"Now ritual is the husk of faith and loyalty, the beginning of confusion."