I was performing BRAF mutation analysis. My sample type is FFPE tissue. Now if you look at the gel pic there are a few samples which did not amplify and all the samples have non specific binding. We are using Hotstar taq (Qiagen). We have done some troubleshooting like changing DNA concentration, using fresh reagents, fresh primer, etc. But we still are unable to get the PCR product without non-specific amplification. Any suggestions will be helpful.
Dear sangeet,
Hello and hope all is well to you.
I saw the picture and the specific band is observed while you have smear and some non-specific bands, also, your specific band is very weak.
here for further assessment i need the following information:
1. please send me the sequence of your primers and i will check it for specificity.
2. Are these primers bind to the mutation?
3. What is the method of DNA extraction?
4. have you ever used a good quality DNA from peripheral DNA with these set of primers?
Yours,
Heidar
Hi,
you could also change the annealing temp. We use 57 C for BRAF. Hotstar taq works well in our hands. Welalso use FFPE, samples up to 20 years old. Works almost always.
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