Kunkel Oligo-Directed Mutagenesis

[Chris Greene]

I have recently started introducing a series of silent mutations into a construct using the Kunkel method of Oligo-directed site directed mutagenesis and would like some advice.  My first attempt worked fine but the efficiency was low.  First of all, instead of using M13, I am using pBluescript
as my cloning vector.  After making single-stranded DNA and carrying out the invitro reactions, the control transformation of 1/10 or the reaction mixture into a Dut- Ung- strain gave plenty of transformants while transformation of the rest into a Dut+ Ung+ strain gave a total of 21 colonies; this is as expected, even if a bit low.  However, out of the colonies from the Dut+ Ung+ strain only 1 had the desired change.  I am wondering if there is any way to increase the efficiency of this method.  If I increase the concentration of Uridine in the culture when isolating ssDNA will I increase the ratio of desired changes?  Or is it possible that increasing the Uridine concentration above 25ug/ml will introduce too many Uridines into the opposite strand, and if so will this make the plasmid non-functional.  Just a thought.

If would greatly appreciate any advice

Chris Greene


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Chris Greene                 <>  The great tragedy of Science-the
cgreene at bimcore.emory.edu    <>  slaying of a beautiful hypothesis
cgreene at unix.cc.emory.edu    <>  by an ugly fact. -- T.H. Huxley
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