Hi, I'm a starting graduate student who's going to experience the world
of microarray.. I'm glad to find this great forum.We are planning to use RNA probes for the hybs with
two step (aaUTP-mediated) labeling. I referred to Pat Brown's
protocol for cDNA 2step labeling, where they indicate to use 4M
Hydroxylamine to quench the coupling (second labeling) reaction.
I consulted Sigma for available hydroxylamine salts, but none of
them is mol. biol. grade, and they suggested that if one
make 0.2M Hydroxylamine. HCl solution, the pH falls around
3.2. Since I am using RNA probes, I get a bit nervous that
Hydroxylamine is not RNase free grade, and consider about
the pH of the quenching solution because it might exceed the
buffering effect of sodium bicarb. Has anybody tried to quench
the reaction with Pat Brown's protocol for RNA probes?
Or do I really have to quench the reaction before re-purify the
RNA anyways (I suppose all the amino groups on UTP are
to be occupied by the time you throw the quencher in, so
wouldn't expect to see any difference in labeling efficiency)?
Any comments will be of great help. Thanks!
Daisuke Hattori
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