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cloning problem


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#1 alex.tina1901

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Posted 22 March 2012 - 11:16 PM

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 45oC but i fail to get an amplification.

Kindly suggest how do i get over it..



awaiting for a solution

thanxPosted Image

#2 leelee

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Posted 23 March 2012 - 12:59 AM

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

#3 phage434

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Posted 23 March 2012 - 04:21 AM

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




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