sequencing inverse PCR

Shahram Mori smori at opus.nmsu.edu
Fri May 27 00:56:43 EST 1994


cobrien at eagle.wesleyan.edu wrote:
: My lab is doing inverse PCR in order to get sequence flanking a P element. We
: were wondering if anyone has tips, references, protocols on directly sequencing
: any products by PCR.
: 						Thanks,
: 						Christine
: 						Wesleyan University
Dear Christine,
There is a great review/ protocol in "Analytical Biochemistry" 216:1-14 1994.
There they talk about basically two different protocols that you can use.

1) Incubate Primer with template ( annealing step) , then extend the
primer by Pol and dNTP's and one Labelled dNTP ( Labelling step). This
gives you oligo's of between 20-80 ntdes. The Final step, extend and
terminate by ddNTP.

2) This one has the primer labelled rather than the nucleotide and allows
you to get the sequence starting from the 3' of the primer. This way you
won't lose potentially large stretches of sequence between the primer and
your labelled nucleotide.

I like the latter better.
Good Luck.
Cheers.
Shahram Mori
Program in Molecular Biology
Dept. of Chemistry
Box 3C NMSU 
Las Cruces NM
88003
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