2 replies to this topic

[alex.tina1901]

hii..
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 45^oC but i fail to get an amplification.

Kindly suggest how do i get over it..



awaiting for a solution

thanx

[leelee]

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

[phage434]

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