Inverse PCR

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Nov 10 12:49:31 EST 1998


In Article <363A8C0E.679C at wam.umd.edu>, ansari at WAM.UMD.EDU (malin) wrote:
>Hi,
>
>I did inverse PCR twice recently but didn't get the PCR product. The
>protocol I used is as follows:
>
>2-4 ug Arabidopsis genomice DNA was digested by Xho I O.N.to the
>completion (detected by agarose gel). The digestion volume is 100
>ul.then 65C 15' to inactivate the enzyme. Ligation rxn is set up by 50
>ul digestion product, 50 ul of 10X ligation buffer, 10 units of T4 DNA
>ligase from BMB and dH2O to the final 500 ul volume. RT 4 hr and then
>65C inactivate the ligase again. 1,2 and 5 ul of ligation products were
>used as template in a 50 ul-volume PCR reaction. They all failed to
>produce any band. I saw nothing on the gel.
>
>Your suggestion and practical protocols are warmly welcomed!

Two suggestions: 1) Use restriction enzymes that recognize 4-base sites,
instead of 6-base sites, to make the amplicon smaller, and 2) Try a variety
of different enzymes in parallel (I've done this before with 8 different
restriction enzymes and got a product with one).

Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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