Site-directed mutagenesis (using PCR)...Help?

[Michael G Abel]

Fellow Bio.netters,

	I am trying to perform site-directed mutatgenesis on my favorite 
gene using the PCR based overlap-extension technique to generate two 
initial fragments of my gene with a 17bp overlap containing a single 
point mutation.  The next step involves the purification of the two PCR 
fragments and using them as the template for a second round of PCR 
amplification with two flanking primers to generate one long PCR fragment 
made up of the combination of the two fragments.  
	I have been successful in producing the two initial PCR fragments 
but have not been able to generate the full length product needed for 
subcloning and expression.
	Has anyone used this technique to introduce point mutations in 
their gene(s) and could you please e-mail me some hints as to what might 
increase my success of getting the full length product amplified?

	Anyone that has used the PCR Megaprimer technique to generate point 
mutations, your comments would be greatly appreciated as well (I have the 
ability to do either technique with the primers I have designed).

Thanks very much for any suggestions and for your time,

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      Michael Gregory Abel      Abel at utk.edu      423-974-2933 (office)
     University of Tennessee   Dept. of Microbiology   423-974-4007 (FAX)
    Knoxville, Tennessee 37996-0845    423-974-4004 (Departmental office)  
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