separation of ds-oligo (21-mer) from ss-oligo (also 21-mer)

OPUS cjones at
Fri Dec 17 17:53:00 EST 1993

>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
 Arnold 703       |
Boston MA 02144   |
617/956-6874      |

More information about the Methods mailing list