What happens to 32P-dCTP when it goes KABLOOEY?
smithwhi at students.msu.edu
Thu Mar 17 22:31:00 EST 1994
In Article <2m89t7$7la at news.u.washington.edu> "wolffie at u.washington.edu" says:
> URGENT. Answer required by Thurs. 3/17.
> So I have all this stale (month-old) alpha-32P dCTP that I have to use
> because They consnabulated our nukes order.
> What happens to an alpha-32P-labelled nucleotide triphosphate when the 32P
> decides it's time to disintegrate? Does it blow completely to smithereens,
> leaving nothing to compete with the remaining 32P-dCTP in the labelling
> reaction, or does it leave some kind of mongrelized pyrophosphate with a
> nasty alpha-sulfate to bung up the works?
> In other words, can I still get high specific activity in my probe by simply
> adding extra 32P-dCTP to the labelling reaction and/or letting it run
> longer, or does the Klenow enzyme utilize the spent 32P-dCTP, giving a
> colder probe?
When the radiolabelled portion of 32P dCTP decays, the recoil breaks the
phosphodiester bond. Additionally, the beta particle usually damages another
dCTP molecule so that Klenow will no longer accept it as substrate. So the
specific activity of the probe is not affected, however the ability to make
probe is severely affected - no substrate has consequence
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