Carbonate buffer hydrolysis of RNA

Ned Mantei mantei at cell.biol.ethz.ch
Wed May 17 00:55:56 EST 2000


In article <13bd218c.072a41f3 at usw-ex0101-007.remarq.com>, Belscotia 
<belscotiaNObeSPAM at yahoo.co.uk.invalid> wrote:

>technique of carbonate buffer
>hydrolysis to shorten a 600nt labelled cRNA probe to about 150nt.
>Basically the pH of the cRNA is adjusted to 10.2 by adding
>Na2CO3/
>NaHCO3 to 0.1M and incubating for about 45 minutes at 60 deg C.
>
>This technique works well but I am wondering if anyone out there
>actually knows *how* it works.

Alkaline hydroylsis of phosphodiester bonds at random. The 2'-hydroxyl 
is ionized and can then engage in nucleophilic attack on the phosphate 
to which the adjacent 3'-hydroxyl is attached. The initial product is a 
2',3'-phosphate and a 5'-OH.
Note that, contrary to a formula in common use, the size of the initial 
product doesn't play a role in determining how long to incubate to get a 
certain size product--longer starting product has more bonds and so will 
have a higher chance of getting a "hit".

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland




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