Hi Fred, If I understand you correctly, you are concentrating your 50uL reaction to 4uL and then transforming all of it. If that is correct, that is WAY too much DNA, especially based on what I see on your gel. Too much DNA inihibits transformations and that is a very common mistake. Assuming I am reading you correctly.
Simply transform say 2.5uL of the PCR reaction (post DpnI) directly into cells and that is likely to work. You could also try, say a bunch of transformations with 1uL, 2.5uL and 5uL. Good luck and I can help you out further to get your mutants! Raji On Fri, Feb 3, 2012 at 12:13 PM, Fred <ccp4bb.l...@gmail.com> wrote: > 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. > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
