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Fecal DNA PCR not working
I am doing genotyping of wild ass fecal DNA.The PCR program I use for amplifying genomic DNA for microsatellite genotyping is:
95 deg celsius, 5 min
then 3 cycles of:
15'' @ 95 deg;
30'' @ 60 deg;
30'' @ 72 deg;
then 3 cycles of the same but with 58 deg instead of 60, then 56....48, then 30 cycles of the same @ 48 deg.
This touchdown program was not developed by me, but was paritally working when I joined the lab. A simple "regular" PCR program does not work for these primers.
In most reactions, only the positive control works (blood DNA), while my samples which are fecal DNA, do not work.
What can I do to improve the PCR conditions to make the fecal DNA work?
Re: Fecal DNA PCR not working
Try to improve your DNA extraction protocol - feces contain many PCR inhibitors and without additional DNA extraction steps PCR doesn't work efficiently, if at all.
Have you checked your DNA purity and amount?
|dna , fecal , pcr , working|
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