Agarose Gel electrophoresis
Posted 07 December 2010 - 10:01 AM
Posted 07 December 2010 - 12:24 PM
Hi, I'm not sure if my reply will help but I'll take a crack at it. I can't tell exactly what's going on without looking at the gel, but perhaps you have incomplete digestion of one or both enzymes? Also, I'd take another look at the sequence to make sure that you're cutting what you think you're cutting. My steps would be something like: 1) resubmit sequence to NEBCutter 2.0 (http://tools.neb.com...tter2/index.php), tell the cutter that the sequence is circular and hit submit, then under main options hit custom digest, enter in your two enzymes, and then also get a profile for each enzyme used individually; 2) rerun the digest this time including a single digest with both enzymes (to make sure that each enzyme is working) and the uncut plasmid for comparison. Those are the steps I would take. I hope I've helped a little.....
Posted 07 December 2010 - 10:14 PM
The only thing that come to my mind is that probably when you ran your digests you used the circular ladder and that is what is giving you higher bands.... You might be getting four bands, because of incomplete digestion by both the restriction enzymes you are using, but I think you could do the digest again, run it on a gel again, making sure your ladder is correct. Hope it turns out well....
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