End labelling a primer

Ravi Ramachandran rravi at u.washington.edu
Fri Mar 20 16:05:03 EST 1998


John Fellers wrote:

> Does anyone have a protocol for end labelling a PCR primer?  Thanks in
> advance.
>
> JPF

  John: I'm assuming this is for 5' end-labeling procedure:
The protocol I use is as :
primer                50 pmol
10X PNK Buffer    2.5 microliters
T4 PNK                0.5 microliter
gamma 32P ATP    20 microliters

Mix well, incubate at 37 C for 30 min; 5C for 15 min
Separate away from unicorporated nucleotides.

I use Boehringer  Mannheim T4 PNK and a G-50 separation colum from
Pharmacia

This does not imply any product endorsements!!!!!!
Cheers,
Ravi
--

************************************************************
Ravi K. Ramachandran
Senior Fellow
D-532, HSB, Box 357470
Department of Pathology
University of Washington
Seattle, WA 98195.

Phone : (206) 543-5363
Fax : (206) 543-3644
email : rravi@ u.washington.edu
Web: http://weber.u.washington.edu/~rravi/

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