Stratagene Site-directed mutagenesis

zxiong at ag.arizona.edu zxiong at ag.arizona.edu
Fri Feb 7 09:15:41 EST 1997


I am trying to use the QuikChange Site-Directed Mutagenesis kit from 
Stratagene to change multple amino acids in a virus sequence. I am trying two 
different primer sets. The first primer set is 66% GC and has a Tm=78.16C. It 
is a 36mer with 12 perfectly matched bases at the 3' and 5' ends. The second 
primer set is 56% GC and has a Tm of 70.7C. It also has 12 perfectly matched 
bases at both ends. I did the thermocycling using the parameters they 
outlined in the manual [(1 cycle 95)16 cycles of 95 for 30s, 55 for 1m, and 68 
for 7 min.] I obtain good results with the contols provided but I get no 
colonies in any of my reactions. I tried varying the amount of template from 5 
to 50 ng like suggested by Stratagene with no success. I have run the PCR 
products in an gel and the only band that shows up is the 4.5kb pWhitescript 
control. I would greatly appreaciate any suggestions from anyone having 
experience using this kit.

Please feel free to respond to me by e-mail at
rlangham at aruba.ccit.arizona.edu

Thank You, 
Rick Langham
Plant Pathology
University of Arizona 



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