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In the end of the PCR process, do you destroy the parental plasmid? If not, it will give you the background. --
Ah, the PCR by mutagenesis problem. The efficiency depends on the methods used. The use of enzymes may also affect the efficiency (I've heard that Vent polymerase is bad for mutagenesis). I would recommend that you use Stratagene Quikchange kit (no affiliation). More expensive no doubt, but you can easily get the enzymes yourself without buying the kit (don't tell Stratagene) and do the mutagenesis reactions. I'm not sure but you might find the protocols on their website. Just need to get two oligos (purified) and you can get your mutants in a day or two. Easy peasy. --
Running pcr mutagenesis with mutagenic oligos. Usually works great. All reactions turning up the right size fragments and colonies grow after transformation of end product. In the end, sequencing shows all wild-type DNAs. Any ideason what is causing this problem? --
Maybe the 2nd round of PCR mutated your muataion back to WT.Try a high fidelity Taq. --
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