I am trying to perform the control PCR protocol with the plasmids provided in the Surveyor kit, but have not been able to get successful amplification. I tried using a PCR buffer-MgCL2 in addition to a 50mM MgCl2 solution. The polymerase I am specifically using is Platinum taq polymerase. Any suggestions would be greatly appreciated.
In my hands, Plat-Taq is not particularly efficient in amplifying difficult amplicons or dealing with crude gDNA preps. My suggestions: 1) Make sure your gDNA is clean and of high-quality. 2) Try regular Taq or any other PCR enzyme that you have in your lab. Finally, when it comes to gel electrophoresis, add no or little dye to your DNA loading buffer, or else your digested bands would be hardly detectable.
Hi
The Surveyor Enzyme used for mismatch cleavage assay. I agree with Kaveh on the point to use good quality DNA. However, if you are only interested in surveyor endonuclease activity, you can try any other known variant sequence amplification as a control test cleavage activity like i used in "COTIP: Cotton TILLING Platform". I used indigenously isolated CEL1 instead of Surveyor But if you only want results from Kit's control PCR, check primer annealing temp. and concentration of control plasmids.
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