tag:blogger.com,1999:blog-32079676.post4743743558862293118..comments2024-02-13T21:22:02.522-08:00Comments on RRResearch: And the USS is bent at...Rosie Redfieldhttp://www.blogger.com/profile/06807912674127645263noreply@blogger.comBlogger1125tag:blogger.com,1999:blog-32079676.post-65749698924235385842012-05-18T16:51:52.634-07:002012-05-18T16:51:52.634-07:00It's probably from the MgCl2 itself. All the s...It's probably from the MgCl2 itself. All the sources I've ever seen regarding migration of DNA in PAGE are based on TBE buffer, without Mg. I know from experience that including Mg substantially reduces DNA migration in the gel.<br /><br />The other possibility is if your acrylamide:bisacrylamide ratio is a lot different than the one in the cloning manual. That can change the degree of crosslinking in the gel and alter migration, but not to the degree you're showing here (at least, not in my experience).<br /><br />You're aware that there's a significant literature on sequence-specific bends in DNA, right? IIRC, having 2-3 consecutive As repeated at ~ 10-11 base intervals (i.e. once per turn) can cause a significant bend.<br /><br />One thing I remember people did to help prove bending is to circularly permute the supposedly bent sequence. That is, imagine putting your presumed bent sequence into a 200-300 bp circular DNA. Now imagine cutting the circle at different places so the base sequence stays the same, but the presumed bent part is at different distances from the end. If it's truly bent, you expect the slowest gel migration when the bend is in the middle. When the bend is at either end, migration should be similar to a random sequence.<br /><br />Apologies if you're already up on all this!rhdurlandnoreply@blogger.com