PAGE purification and analysis

yan sun via methods%40net.bio.net (by yansun2005 from gmail.com)
Sun Apr 6 19:15:14 EST 2008


Dear all,
I was confused  by my PAGE gel purification for a while.
I tried to purified spin labeled DNA and non labeled DNA through 20%
polyacrylamide gel. My DNA is around 8,200 dalton, the attached spin label
material MW is around 200g/mole, which means I try to separate two DNA
bands, the molecular weight difference is 200 dalton. My DNA is 27 mer.
The problem is that two bands are overlapped. the band isn't sharp, the
resolution is not good, the band is in meniscus shape. wasn't straight at
all:(
My lab mate who successfully purified this two band  get very sharp and
clear bands.I "exactly" follow her protocol. but do not get the satisfied
result:(
I used constant power, 15w.
my guess is the temperature is too high, should I run the gel in the cold
room? but I pretty sure she run it in RT.
I pour 25ml acrylamide solution, should I degas it first? my 10% APS is new,
TEMED is good. is that because the poor gel quality?
but she didn't degas the gel. I know it really depends on individual hand-on
experience.
I don't really know how to improve it, how to get sharp and clear bands?
Anybody has suggestion?
Thx a lot!

-- 
Y. F.S.


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