Poor resolution when isolating ssDNA?
(by evan.g.reed from gmail.com)
Thu Jun 12 16:56:35 EST 2008
I am trying to isolate ssDNA after binding dsDNA oligos with
biotinylated antisense strands to strep microbeads, denaturing with
NaOH, then adding the supernatant containing the sense ssDNA to a
NAP-5 sephadex desalting column. Unfortunately, I am getting terrible
resolution when I try to elute on the column -- even after 2 mL of
elution, I am still getting detectable levels of ssDNA in the eluate
(dsDNA oligos of the same length elute just fine in 0.5 mL, with
acceptable resolution). The amount of DNA loaded into the column
should be well below the binding capacity so I am not sure why this is
happening. Has anyone done this before? Is there a better way to
purify the ssDNA instead of this column?
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