I´m going to analyze my soil N genes with a ddPCR. The qPCR don´t work with my samples, due to inhibtor problems. Is it possible to use Primers with a fragment size of 600 bp with a ddPCR. Which program do you use for long fragments? someone has experience?
Thanks a lot
Wishes Evi
hello we have just done a thorough comparison between qPCR versus ddPCR (I pasted the reference below, its open access). First as a general guideline I would use a smaller amplicon, basically our amplicons for ddPCR were the same as for qPCR. In the publication we do not use soil DNA but we do use it a lot in the lab and we perform a aluminium sulfate precipitation which enhaces our PCR yield a lot. But if it doesnt amplify with q it will not work with dd. Try solving your inhibitor problem first, the power soil kit from Mobio works well, but even with this one we do the aluminium sulfate precipitation step.
Here is our dd vers qPCR comparison.
Taylor, S., Laperriere, G., Germain, H. (2017). Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Nature Scientific Reports 7, article number 2409.
I also attached the aluminium ppt protocol.
Good luck
Thanks a lot for your answer..I can´t use a new protocol for extraction, because we don`t have fresh soil for a new extraction :-/.
I tried to purify my DNA with QIAquick Purification Kit but it didn´t work well - i lost a lot of my DNA and qPCR still does not work. Do you have an other idee, how i can purify my extracted DNA?
Thank U
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