dna separation in native PAGE - (Jun/03/2009 )
im trying 2 do separation of nucleic acids by native page. i have 2 resolve by restriction digestion products that are having base pair difference of 56bps.but the fragments size is around 2000bp/
i should choose acrylamide concentration according 2 the resolution i want ie 56bp difference 2 b obserevvd in gel or according to the size of the fragments ie 2000bp.
im confused ??can any1 help plzzz
2000 bp is approaching the upper limits of resolution for acrylamide gels. You will need a quite low percent gel -have a look at Roche LabFAQs for the percent, and run with TBE.
If you want to see only 56bP on the gel then its fine but if you want to see both 2000bP and 56bP, I don't think it'll be possible on acrylamide gel. why don't you try to run a higher percent (2-3%) agarose gel, probably you'll be able to see both the bands.
You should also check out:
1. Brody, J. R. and S. E. Kern. 2004. Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques 36:214-216
2. Brody, J. R., E. S. Calhoun, E. Gallmeier, T. D. Creavalle, and S. E. Kern. 2004. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. BioTechniques 37:598-602.
SB gels are very good at resolving small size differences (see especially reference 2).