I am trying to amplify 3.1kb fragment from genomic DNA but i dont get anything at all in Gradient PCR above 60C. But when i lower the gradient from 48 to 60C i get huge non-specific amplification not my band of interest.
I tried to amplify it from cDNA doing gradient from 60 to 70C i get amplification at 1.5kb (corresponding to a transcriptional variant of my gene which also includes binding site for my primers) not at 3.1.
however in case of genomic DNA i neither get 1.5 kb nor 3.1 kb at 60C can any one suggest me a possible way out
I think for one thing if you would like to get genomic DNA, you should use DNA as template , not cDNA. And then you can try more annealing temperatures, for example, from 50-60C set 5 conditions. I suggest you to use High fidelity enzyme . And try different kinds of enzymes if you have access to them. I have done with 6-7K fragment amplification.
Thanku Yunjing Ma i am already using a high fidelity enzyme Q5 from neb also i did gradient PCR of annealing from 48C to 70C. i didnt amplification of my gene of interest but i got non specific amplification at 58C. however when i used cDNA as my template i got amplification of transcript at 1.5kb which corresponds to a transcript variant of my gene but not my gene of interest.
Hi
Would try a touchdown PCR. Are you sure about your DNA quality and is your protocol relaxed enough meaning enough extension time?
Sven
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