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dna separation in native PAGE


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#1 deespike

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Posted 03 June 2009 - 10:29 AM

hi all

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

#2 bob1

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Posted 03 June 2009 - 04:02 PM

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.

#3 neuron

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Posted 06 June 2009 - 04:11 AM

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.

#4 HomeBrew

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Posted 06 June 2009 - 09:23 AM

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).




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