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Problem restriction digest on single fibers

restriction digest

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#1 KimV

KimV

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Posted 23 May 2012 - 10:59 AM

Hello,
I'm trying to perform a restriction digest on single fibers. I'm using 2 enzymes BanII and Cviki-1. What I do is I cut 1 fiber out of muscle tissue on a slide (LCM) and perform than a dna extraction. Then i do a PCR with a fam labbeled primer. In the next step I do the restriction digest. The problem I experience is that not the whole sample is digested. about 10% stays undigested. I tried already using different restriction buffers, adding more enzyme, longer incubation times, adding BSA but nothing seems to help.
If I start with 200 ng DNA in a pcr reaction and perform than the restriction digest everything is digested. So I assume it has something to do with the small amount of DNA.
Does anybody have experience with restrction digest starting from small amounts of DNA?





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