I am having problems with the Kunkel reaction, yield is very low and product I am getting is not pure.
The product of a Kunkel reaction is usually not completely pure. Apart from the remaining single stranded DNA, you usually get 3 bands if you run the product on agarose gel. The one running lowest is the correct product. The one above the correct band containes non-ligated product. The top band is a strand-displaced product. (See this pic: http://www.springerimages.com/Images/Biomedicine/1-10.1007_978-1-61779-974-7_10-0) If the lowest band (the correct product, not the remaining single stranded DNA) is well visible, than your reaction has gone well.
The possible reasons for the failure of the mutagenesis are numerous. Maybe the annealing part of your oligonucleotide is not long enough (I use around 18 bases on both sides of the region to be mutated, even if I only want to introduce a single mutation). Maybe the enzymes you are using are not working properly. I also suggest that you use freshly isolated single stranded template.
Hope that helps!
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