bsh at MED.PITT.EDU
Wed Feb 10 18:02:30 EST 1993
> After gamma-labeling a purified (OPC) oligonucleotide, I could observe
> two distinct bands on a 20 percent PAGE.They show a migration behaviour
> as if they would differ in a single terminal phosphate group.
> Dephosphorylation with polynucleotide kinase
> (3`phosphatase) did not result in one distinct band.This would indicate that
> the problem is not an additional phosphate group.
The rationale that we have been using in analyzing electrophoretic
behavior of gamma-labeled oligos goes like this. May be someone could
expand or contradict.
The important feature we look for is that 90% or more incoroporation
in the single fragment indicates a good synthesis.
The smaller bands correspond to partially synthesized products,
although they should not be taking up any label on the basis of nature
of the chemistry involved in blocking all the groups.
The larger bands correspond to those oligos from which the protective
groups have not been completely cleaved, accounting for the increase in size.
the above explanation also holds good here.
Sometimes we have seen bands that are not supposed to be there.
Such as one to three smaller bands just below the presumed oligo. But
almost all the specific (as opposed to degenerate ones) oligos have
worked quite well in PCR and sequencing. I would be interested to
know other's experience. For this kind of analysis, I always use the
sequencing gel and its conditions.
bsh at med.pitt.edu
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