I am trying to amplify the full length human mtDNA(~17kb) from single cell for NGS. Currently, I am using the long range PCR strategy by NEB Q5 polymerase. I can get the mtDNA when starting with regular gDNA template amout(~20ng), but I can hardly get any bands with 10-fold diluted template which is still 100-fold concentrated comparing to single cell system. I tried to increase the cycle number, but it didn't seem to work. What is the optimal condition for the single cell LR-PCR? Does Multiple displacement amplification (MDA) work for this purpose?
Hi Yital
MDA is a good method to amplify DNA, simple and robust. but on the other hand it amplify all the DNA, and mitDNA just represent about 1/600.000 of the whole DNA. after MDA amplification, you'll need to enrich the sample by capturing or amplifying specifically the mitDNA (sureselect, ampliseq, truseq..) before doing your library.
fred
visit this link.
http://www.sciencedirect.com/science/article/pii/S0006291X85716488?via%3Dihub
have a good work .
visit this link.
http://www.sciencedirect.com/science/article/pii/S0006291X85716488?via%3Dihub
have a good work .
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