Posted 08 October 2004 - 04:50 AM
Posted 09 October 2004 - 10:23 PM
I mean you must make sure that you have choose the right plasmid, primers will not anneal to the wrong template no matter how hard you've tried.
Posted 10 October 2004 - 08:08 AM
Posted 10 October 2004 - 11:01 PM
Any other ideas ??
Posted 12 October 2004 - 05:29 AM
The promoter region that i've been working on is also very AT rich and i've never had a problem. It could be worth making sure that your oligos are fairly long (>25?) in order to get decent Tm's.
Bioline also make a good polymerase called BioXact, which has worked for me in the past.
Both of these are high fidelity. I am using Expand for site directed mutagenesis of an AT rich region without any problems.
If you are worried about secondary structures, you could always linearise your plasmid template?
Posted 04 March 2005 - 07:35 PM
I never used Pfu enzyme. But this is the first time i will be using this Pfu to amplify promoter fragments of 2kb. I know pfu is very costly to use. so iwant to standerdize the conditions by using taq polymerase enzyme.Any body has an idea about the difference in annealing temparatures for Pfu and Taq polymerase.
Thanks a lot