Mutagenesis problems

[Karen Kroeger]

Hi,

I have been uisng the Clontech Trnasformer-Mutagenesis Kit to introduce a 
6 base pair mutation.  In the kit a mutagenic oligo is used. I have an 
oligo 30bp in length, which contains 12bp matched either side of the 6 
mis-matched base pair.  I have used to kit according to the protocol and 
have done it several times, but have not had any success. I have tried 
varying the amount of mutagenic oligo used, but to no effect.  I think 
the mutagenic oligo may contain  a secondary structure which is 
preventing it from annealing to the plasmid.  Does anyone have any 
suggestions or changes to the standard protocol that will allow me to get 
around this problem.  Or anyone that has had problems using the kit to 
introduce this many base pair changes.  Any help or suggestions would be 
great. Reply by email.

Thanks
 Karen Kroeger
PhD Student
Department of Biochemistry
University of Western Australia
Perth, Australia
Email : karenmk at uniwa.uwa.edu.au