Hi, 1) anneal your PCR product (after dpn) 2) use smaller amount of material to transform, as already pointed out 3) if you could share the primer design, maybe we can find something in there
Artem On Fri, Feb 3, 2012 at 11:13 AM, 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.
