Acrylamide gel to resolve DNA of similar size? - a little advise please (Dec/06/2005 )
hi! i have to resolve bands of dna that are kinda similar, so i was thinking about usuing acrylamide, but the range of the smallest and largest band are 650 and 2200, is that a problem?
let me make things clearer... i needto separate and resolve bands that are 650, 750, 900, 1000, 1350, (upto here no problem), 1750, 1800, 2150 and 2200 bp. cant get them in separate tubes. is acrylamide possible?
sure ... but agarose should also resolve these differences. why not use, let's say, 1,2% agarose?
only problem will be 2150 and 2200. to resolve these better use 0,7% agarose
i have been using agaroseat .7% but when i run it too long, the bands get really weak, how do i overcome that? if i dont, i dont get a good resolution. any tirck?
metaphor agarose is good and is an alturnative to acrylamide gels to resolve at 2bp resolution, the bands you would like to detect can be resolved on normal agarose,
the faint bands on your gel could mean that your ethidium bromide has leached from the gel during the run therefore you have faint bands, you may want to restain your gel after the run and rinse with water and you should find that your bands are now bright again.
what voltage you run your gels? higher voltage leads to more defined bands in my hands. using 0.5xTBE i can go up to 20 volts/cm.
it is possible resolve them in agarose gel(1.5%) prepared on TBE buffer, as the gel will be thick you have to be little careful...also run your gel in voltage for longer time
hope you get the result
try agarose gel(7% or 8%) prepared on 0,5 TBE buffer, ~300v ~14-16h
depending on the gel size, i'd say, a bit long and a bit high voltage ... but as i said, depending on the gel size, that's why voltages should be always put as V/cm