Inverse PCR

malin ansari at WAM.UMD.EDU
Tue Nov 10 12:05:52 EST 1998


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!

Thanks!

Black
cxwang at wam.umd.edu



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