PCR-PROPDUCT PURIFICATION

Charlie Wright Genetics cw117 at mole.bio.cam.ac.uk
Mon Nov 21 06:06:43 EST 1994


mbjjb at s-crim1.dl.ac.uk (J.J. Baker) writes:

>sl6dp at cc.usu.edu wrote:
>: Going through the literature I have found that it is possible to ream-
>: plify PCR products directly from the band after selection on a low-melting
>: agarose gel. I am not sure if I understand it: it is really possible
>: to run the PCR with a portion af the agarose band, without even
>: taking the DNA out????. Anyaway, which is a good method for PCR product
>: purification if I want to run a secon PCR with another primer?, I mean
>: no comercial kits...Thanks

>Yes it is! Though I can't remember the exact referencefor this technique,
>this is the method I use; I pierce the bands of the gel with a wide bore
>needle and then put the needle into the reaction buffer for PCR then leave for 
>about ten minutes or the length of your tea break at room temperature
>Then simply take out the needles, add your Taq and run for 5 more cycles
>than usual.

>So far this method has only failed me once and that was with a very faint
>band so obviously the amount you load on the gel in the first place is
>quite important.

>Trust me, it works!

>Jon Baker

>Centre for Molecular Biology
>Dept Chemistry
>The School of Pharmacy
>29/39 Brunswick Sq
>London
>WC1N 1AX   UK

A chap in our lab also uses this technique.  I believe he actually blows 
the plug of agarose (low melt) into the eppendorf tube and then adds the 
reagents for PCR.  You'll find that the lmp agarose will melt quite 
readily at 96c and then it will be too dilute to cause a problem, usually.

Charlie
-- 
C. R. Wright                                    Dept. of Genetics
+44 (0)223 333970 telephone                     Univ. of Cambridge
+44 (0)223 333992 telefax                       Downing Street, Cambs.
cw117 at mole.bio.cam.ac.uk                        CB2 3EH, England



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