Two questions

Ted Michelini tedm at darkwing.uoregon.edu
Tue Sep 19 11:37:38 EST 1995


In article <43mo3e$734 at portal.gmu.edu>, isagdeev at osf1.gmu.edu (Igor R
Sagdeev) wrote:

> Dear netters,
> 
> I have two questions for those who have any experience in these two
> things:
> 
> 1. What are the easiest and most efficient ways of getting rid of
>    unincorporated primers and nucleotides after a PCR without losing
>     too much of the product?
> 
> 2. what is the simplest protocol for TCA or HCl precipitation to
>    assess incorporation of label in generation of radiolabeled
>    probes?
> 
>                                                 Yours, Igor

Igor,
   What I do:

1. microcon30 PCR sample with three short spins and washes with buffer of
choice, note that larger template DNA will still be present. (If one
doesn't want Taq pol around, a protienase K treatment followed by 2-3
phenol/CHCl3 extractions is helpful.)

2. spin column through sephadex G50 after kinase/fill in, followed by a
count on a high dilution. The spin column is needed anyway and the count
efficiency can be pretty accurate if one generates quench curves.

These methods are not mutually exclusive, and the Southern jocks may have
far more accurate ways to quantify probes, probably not as easy, though...

regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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