Dear CCP4users biologists,
I'm trying to make a single aa mutant of a 5.7 kb non commercial vector
with the Agilent's Quick Change Site-Directed Mutagenesis Kit. I have
strictly followed the instructions manual, however, I could not be able
to transform bacterial cells with my PCR product. I can observe the
amplified PCR product before and after DpnI digestion (see image in
http://ompldr.org/vY2x3aw), but cannot get any colony on LB plates. I'm
using very fresh super competent cells so that I've got dozens of
colonies with 60 ng of the parental/non-mutated vector as positive
control. The bands in the referenced image corresponds to 2.5 microL of
a 50 microL reaction volume. I usually concentrate it to 4 microL before
transformation. Also, I've already optimized the primer's temperature
annealing (best is 62 oC) and I've increased the extension time up to 9
min. Is there anything else I can try?
Any help is appreciated!
Regards.
Fred
P.S.: Agilent's e-mail support is not working.
P.P.S.: this might not be of other's interest, address the answers,
please, to my e-mail only.
