PCR inhibition in FFPE DNA - (May/09/2008 )
I've finally managed to extract a good amount of genomic DNA (~100 ng/ul of dsDNA) from formalin-fixed, paraffin-embedded (FFPE) tissue (liver, kidney sarcoma and colon). I used heat and protease in lysis buffer to digest the tissue and then isolated the DNA by phenol/chl extraction and EtOH precipitation.
I ran the DNA on a gel and as expected, the majority of fragments are less than 1 kb, but the PCR products I'm looking for are all less than 500 bp.
Any ideas on ways to clean up this DNA and remove PCR inhibitors? Has anyone tried any of the kits that claim to do this?
I greatly appreciate any advice and suggestions!!!
Dilution is the best and easiest way. You need only a few molecules of the correct template. You might also want to look at the new NEB product, Pre-PCR, which has a set of enzymes designed to repair damaged DNA (nicks, missing bases, oxidized guanine, dU etc.).