oligo deprotection

Paul Morrison morrison at FARBER.HARVARD.EDU
Sun Jun 5 17:25:07 EST 1994


arosenth at crc.ac.uk (Dr. A. Rosenthal) writes:

>Hi everybody,
>somebody just told me that he had heard, one could deprotect oligos by boil=
ing
>them
>for a short while!? rather than the 5hour or o/n 55'C waterbath.
>Any comments greately appreciated.
>Thanks.

Dr. A,
sure for those run of the mill PCR or sequencing primers: 80=B0C , 15 minute=
s
in a screw cap epp. Make sure it's the kind with a black "O" ring. You need
the high pressure but one has to watch out with ammonium hydroxide under
high pressure. The glass vials _will_ explode.  I'm sure you will get some
responses that say you can skip the deprotection.
But if on the other hand the oligo is going into a construction or is a
30mer or longer then I think it's worth the trouble to 4 hr 55=B0C treat it,
dry it down and repeat the deprotection. Sounds like overkill but
incomplete deprotection followed by the e. coli repair system not knowing
what to do with a protecting group seems to be as good an explanation as
any for a deletion in a construction with 100 mers. I hate it when that
happens,   hope this helps -Paul

Paul Morrison   Dana-1030
Molecular Biology Core Facility
Dana-Farber Cancer Institute
44 Binney Street
Boston, MA  02115






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