What's the highest DNA concentration for PCR? - before being inhibited? (Nov/23/2010 )
The title says it all. Does anyone knows the range of DNA concentration where you'd start getting smears and/or inhibition? Is it in the ng or ug range?
Thank you and Happy Thanksgiving.
M.
Depends on the type of DNA. Roche Lab FAQs says it is 500 ng for human genomic, 10 ng for bacterial genomic and 500 for plasmid.
and it depends also on how specific your primers are and how stringent are the PCR conditions.
over 1 ug of both DNA and RNA gives me smear always.
why is it different between human and bacteria?
bacterial genome is much smaller, so 10 ng has more copies of each gene is my guess.
You can easily do cycle titration to test for your own sample. You can remove some DNA sample every 5 cycle during PCR and run on the gel. Good luck.
http://www.bioprotocols.info/index.php
bob1 on Wed Nov 24 22:29:11 2010 said:
bacterial genome is much smaller, so 10 ng has more copies of each gene is my guess.
I though the inhibition was caused by a tube "too crowded" where the primers and enzymes can't find their target.