UPDATE: Removing Unincorporated Nucleotides

kim at m44.unm.edu kim at m44.unm.edu
Tue May 26 16:29:42 EST 1992


Hello Group:

Thanks for the lively discussion on the merits of removal of unincorporated
nucleotides from probe synthesis reactions before hybridization.  It looks like
most people do remove them before using the probe, but there is no real
consensus as to why.

When I make a probe, I like to determine the number of incorporated counts by
TCA or HCl precipitation onto a glass filter, per Current Protocols, so I
don't need to remove unincorporated counts from the entire probe batch if it
won't make a difference in my hybridization reaction.  I tried a comparison of
probes cleaned up with a BioGel P-30 spin column (made in a 0.5 ml Eppendorf 
tube) and an aliquot of the same probe with an equivalent number of
precipitable counts, but without cleanup.  I noticed a slightly
higher background in the blot with the "uncleaned" probe, although this may be
due to another aspect of the test (I also wanted to see if I needed to
prehybridize the blot).  So it looks, at least in this instance, that probe
cleanup was not necessary.  Results from the acid precipitation give me only a
45% rate of incorporation of the radioactive nucleotide into the synthesized
RNA probe.  I'll try this again a few more times before I'd actually say that
probe cleanup is unnecessary, but it didn't seem to hurt this time.

Daniel Kim



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