Ambion HybSpeed RNase protection assay kit

Daniel Chagnovich dchagno at merle.acns.nwu.edu
Wed May 24 12:53:09 EST 1995


In article <3ptjgb$is4 at lyra.csx.cam.ac.uk>,
Bernard Khoo (WCI) <bcek1 at mole.bio.cam.ac.uk> wrote:
>Dear all,
>
>A leaflet from Ambion just popped into my mailbox this morning... it advertises
>a new-and-improved version of their RPA kit, called HybSpeed. Supposedly
>this allows you to complete hybridisation within 10 minutes at 68oC instead of
>a standard O/N hybridisation at 45oC and is 1.5-3 times more sensitive than
>their RPA II kit. Only catch, of course, is the price -- UK pounds 260 for
>100 reactions.
>
>Sounds like they've modified the hybridisation buffer to me; in the article
>they say that it does not involve using a 'crowder' like dextran sulphate, nor
>does it involve phenol emulsion reassociation. Has anybody out there tried this
>kit, or have any idea what Ambion might have done to modify the hyb buffer?
>
>Thanks.

Bernard,

	I have never used the Ambion kit before, I have however performed the
hyb step of RPA in 1 hr at 65o many times before. I first read about doing
this in a paper by Brewer and Ross (Meth in Enzymology 181:202-209 1990.) and
was a bit surprised that they performed their hyb at such high temps for such
short periods of time in tyhe ABSENCE of formamide. I wasn't sure this would
work, so I ran a formamide and temp curve and found that for my probe
(~300nt) 65o for 1hr in 0% formamide worked well. Hope this helps.

Danny



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