Separation of DNA bands of sizes 8.86 kb and 9.5 kb - (Dec/19/2007 )
I need to separate a linearized plasmid 8.86 kb from one 9.51 kb. I am currently trying a pulse field electrophoresis, but have been told that regular agarose electrophoresis is possible, as well as polyacrylamide. If anyone has done this before, please tell me percentage of gel, run time, gel size ,etc that i should try.
Thanks in advance
Are you gel extracting, or just trying to visualize the difference? If visualizing, I'll be a normal gel will just work. You might want to run it longer to position the DNA near the end of the gel, but even that is probably not necessary. If these are related plasmids, you can probably cut the plasmid with some enzyme to reduce the size of fragments and accentuate the size difference of the relevant fragment. For gel isolation, you may need higher separation than can easily be done with a simple gel. You're far away from needing pulsed field gels.
I am doing a partial digest, then gel extracting. Basically I want to replace a promoter in my plasmid, but there are no unique sites. The best site i can find has 3 cut sites, and both drop out fragments are similar in size (one fragment being the promoter i want to replace, the other fragment being my gene of interest). So i have a mixture in my tube of the desired digest, and also the other one, with the difference of the backbone being 650 bp. I cannot cut the plasmids any further because the smaller (8.8kb) fragment is the one i want to extract.
why don't you cut the bigger fragment which is not needed by you with restriction enzymes whose sites are not present in the fragment of your interest?