difficulty about cloning 3.5kb PCR product - (Apr/05/2005 )

Dear Everyone, is there anyone could give me some advice for my long PCR product cloning.

I use TOPO TA kit from invitrogen, it works fine with my 500bp and 1.1kb PCR product. But not for 3.5kb. I did at the same time with another gene, there are no positive clone on the LB plate. Only once I am sucessful, but after sequencing, I lost 400 bp fragment, so I have to start from beginning. But I tried several times, i still could not get any colony.

Thank you!

heather

hi, i have in the past routinely cloned pcr fragments up to 6kb without any problems and high efficiency, so for sure it's not due to topo cloning. however, i always used fresh pcr products and in case they were sitting for several weeks in the freezer, i usually ran a "pcr rxn" with Taq and dATP only to add the required As to the ends of the pcr products. this also holds true if you had used a proof reading polymerase such as pfu in the first place. another thing of course is to check if your competent cells are still okay. cheers.