I am trying to amplify AT-rich promoter with Taq polymerase. Have tried various approaches like varying MgCl2 concentration, changing annealing temperature, changed genomic DNA and primer concentration. Finally, I got non-specific amplification along with the desired bands. But I am not able to amplify again. Even reamplification with the eluted PCR product doesn't work. Any suggestions, as to what may be going wrong?
The following links may provide you certain valuable informations:-
1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145803/
2. http://nar.oxfordjournals.org/content/24/8/1574.full
Hi, who designed the primers? Do you think your primer pair is working properly? Cheers, Nadine
Yes, the primers have been designed very carefully by my senior. I have used it and got the results (non-specific amplification) on two different PCR machines, but I am not able to replicate the PCR again. I thought maybe its a handling error. So I repeated it very carefully. Even asked my senior to perform the same PCR, but we got no results. Even the re-amplification is not working. I have ruled out handling errors as a factor. Have checked the stability of the primers. Yes, but as the last option I am designing new primers also.
If your positive PCR control didnt give you a product when visualized on an agarose gel, consider getting new set of Taq polymerase. I have the same problem before, when changed everything; harvest fresh DNA, new PCR mix, new primers, they worked.
What's the length of your PCR product? Can you tell me (us) your PCR program and your reaction setup?
The following links may provide you certain valuable informations:-
1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145803/
2. http://nar.oxfordjournals.org/content/24/8/1574.full
Sandip, thank you very much. I came here because I have the same problem. I will try that.
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