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question about AmpliTaq - (Feb/23/2005 )

I have a question about using AmpliTaq polymerase in PCR , and then using the digested PCR products in ligation into a properly digested vector. Let me first say that I do a lot of site directed mutatgenesis using the PCR overlap extension method in our lab, and have been using Taq with a lot of success (I know it does not have proofreading ability like others, but since it has not been a problem for me I just kept using it , and it is less expensive than other enzymes.) All of a sudden now I am having problems--specifically, my sequenced clones have the desired mutation I want (that I introduced with my mutated primres) but there are also some (1-2) PCR generated mutations as well. Usually the mutation is from a G or C to A or T that changes the amino acid. These mutations have been random..not always at the same spot. My first guess would be to use a more robust enzyme, but my question is why all of a sudden I am having a problem now? Also, my PCR geneated inserts are not that long...700 bp at the MOST. Any insight, and ideas on what to do next would be great.



My first impression is that you have being riding your luck with Amplitaq for some time. However, some things will alter the ability of the Taq to amplicate accurately, one of the key things is the quantity of dNTPs. What I think might happen is that in the shorter PCR the dNTPs are in far excess compared to the PCR mutagenisis and therefore are more likely to misincorporate. Therefore you could try diluting back your dNTP concentration, however, I would try a mix of a proof reader and Taq that way you get the Taq ease of use but you also get proof reading ability. Rember, the cost of repeating experiments is more than the cost of doing right the first time.




You are probably right about "riding my luck" with Taq. I am in the process of testing another polymerase with proofreading ability. But thanks for your post and input!