separation of ds-oligo (21-mer) from ss-oligo (also 21-mer)
cjones at opal.tufts.edu
Fri Dec 17 17:53:00 EST 1993
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.
>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.
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