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cloning problem - (Mar/23/2012 )

I am trying to amplify a 3 kb cDNA. the genome of my interest is AT rich (77%).I tried amplifying it using Pfu polymerase,incresed Mg conc upto 3mM, decreased the extension temperature to 68C,I reduced the annealing temperature to as low as 45oC but i fail to get an amplification.

Kindly suggest how do i get over it..

awaiting for a solution



How long is your extension time? Can you write out your program for us?


Lower the extension temperature to 63, and make the extension time longer. Some high AT constructs will not extend at 68.