Restriction Enzyme Troubleshooting Help? - (Jun/13/2008 )

Hi all,

I am trying to troubleshoot a problem I'm having.

I have isolated bacterial genomic DNA and did subsequent digests on four samples with Sau 3A, Eco RI, BamHI and Not I (not combined, each sample only got one). I incubated the digests at 37C for one hour. I don't think anyone else has had this problem. I used the appropiate buffers, 2uL for 1ul of restriction enzyme in 17ul of DNA sample.

When I run these four sample on a gel, alongside my orginal genomic DNA sample, I get four large smears at the bottom of the gel and no bands for the digest.

I do get a decent single band for the genomic DNA with not RNA contanimation in that particular well.

The restriction enzymes look like RNA I guess...? I'm not really sure. I don't have much experience running gels... I'm a newbie. At any rate, the smears are in the low MW range. There are absolutely no visible bands in the restriction enzyme digest.

I don't think anyone else in the lab has had a problem with the restriction enzymes at all. I think the buffers were new when I grabbed them.

What could I have done?

Thanks guys

I'd guess you are digesting way too much DNA. What is your DNA concentration? You should digest 500 ng in a 20 ul volume, perhaps less.

Hmmm.... it was 75ng/uL...

I repurified more genomic DNA using a different protocol. It ran well using the same method as above... and was even more concentrated that the previous sample (at least three times more). It came out nicely. I'm wondering, I have heard about trace nulcease activity after genomic purification which can interfer with restriction digests... could this be the case? I extracted with phenol the first time it came out poorly, but I think that removes any possible nuclease activity right?
Thanks again.