separation of ds-oligo (21-mer) from ss-oligo (also 21-mer)
ingrid at helios.anu.edu.au
Tue Dec 21 01:52:23 EST 1993
This is indirectly related, but if you silver-stain an acrylamide gel,
the double-stranded DNA will be black and the single-stranded DNA more
a reddish colour. As Opus pointed out, glycerol is great for changing
the relative mobility of single and double-stranded DNA, but it seems
to vary a great deal whether it's the single or double stranded fragments
that run faster, hence the silver staining test.
In article <17DEC199318533382 at opal.tufts.edu>, cjones at opal.tufts.edu (OPUS) writes:
|> In article <01H6IO3CW7F4BQ0VOW at WISTB.WISTAR.UPENN.EDU>, WILLARD at MVAX.WISTAR.UPENN.EDU writes...
|> >I trying to crystallize a DNA binding protein with its oligonucleotide
|> >binding site. I have annealed two complementary single stranded 21-mers
|> >with a one base pair overhang on each end of the annealed product (A and T).
|> >The ss-oligos were HPLC purified prior to annealing.
|> >When I run a 20% PAGE gel on the annealed product, I see two bands: one
|> >is the more heavily staining band and the other (about 3-4 times less intense)
|> >is of lower molecular weight.
|> >My question is twofold: Does the lower MW band correspond to excess ss-oligo
|> >from the annealing reaction and, if so, how can I separate the ss from ds
|> >Is there some sort of nitrocellulose filter that I can use such that the ss
|> >DNA will stick and the ds DNA will go through the membrane?
|> >Any advice would be a great help.
|> >Thank you,
|> >Rob Willard
|> >The Wistar Institute/University of Pennsylvania Chemistry Department
|> i have been purifying a 120 BP frag from PCR and i see lots of single stranded
|> contamination of my fragment. there is a article by maniatas (in biochemistry, i
|> but can't find the ref) that gives the relative mobility of SS & DS DNA in
|> a variety of gel concentrations. I have successfully used a 10% acrylamide gel
|> with 5% glycerol in 0.5X TBE to separate the ss & ds species. I cut the bands
|> out of the gel, crush them with a glass rod, and elute with oligo extraction
|> buffer (Maniatas). I get ~90% recovery of my fragment.
|> If you are staining your gels with EtBr, then the heavily stained band is
|> probably ds DNA, and the lighter is ss.
|> good luck
|> clyde jones
|> Clyde Jones | "There ain't no devil,
|> Tufts University | Just God when he's drunk"
|> 136 Harrison Ave | -Tom Waits
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