single-stranded PCR

rybicki at uctvax.uct.ac.za rybicki at uctvax.uct.ac.za
Thu Apr 9 09:39:44 EST 1992


In article <30MAR199215501948 at rigel.tamu.edu>, msw1633 at rigel.tamu.edu (Mark S. Whitsitt, N5RJF) writes:
> In article <1992Mar30.142854.9212 at news2.cis.umn.edu>, ernest at lenti.med.umn.edu (Ernest Retzel (1535 49118)) writes...
>> 
>>Why not put a biotin on one of your primers, and fish it out after the
>>PCR with magnetic beads?  This has been done for a variety of situations
>>already.
>> 
>>Ernest Retzel
>>Dept. of Microbiology
>>Univ. of Minnesota
> 
> How about a reference or two on that.  I am not sure about how to biotinylate 
> the primer, ie how many biotins per primer, how much does the biotin affect the 
> PCR reaction , and how do you determine how many/much of the magnetic particles 
> do you add per estimated biotin content?
> 

Basically, you ask whoever is doing your oligos to use "biotin-on
phosphoramidite" chemistry: this ensures you get a biotin wherever you want one
in the sequence, but one should place it at the 5' end, where the odds are it
will not interfere with the annealing or with the extension, and which means
you can fish it out with any avidin/streptavidin conjugate, whether on a
magnetic bead, or column material.  Refs: none offhand, but any oligo-maker or
other PCR freak should have a bundle.  Also any archiving programme / online
literature database should be able to pull out a ton.

Ebnjoy

Ed Rybicki
Dept Microbiol, Univ Cape Town



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