degenerate PCR

Dr Koen A.L. De Smet k.desmet at ic.ac.uk
Fri Feb 28 04:43:05 EST 1997


I have the sequence of a gene of two related bacteria. I would now 
like to set up PCRs to amplify this gene from other species in the 
genus, and then sequence the PCR product. Can anybody give any tips on 
degenerate PCR. Advise on the following questions are much 
appreciated:

1. How much redundancy is still acceptable in the primers?
2. Should I use all possible codons, or allow for mismatches, to 
minimise redundancy?
3. Should I increase primer concentration in the PCR?
4. Any tips on annealing conditions?
5. I intend to use these degenerate oligos as sequencing primers as 
well (which works fine for specific oligos), should I use more primer 
than in standard reacions?

Any help, tips or advise muc happreciated!

Koen
 

_______________________________________________________________

        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain
                                     
        Tel: (+44)-(0)171-594 3946  
        Fax: (+44)-(0)171-262-6299   
        Email: k.desmet at sm.ic.ac.uk
	       http://www.sm.ic.ac.uk/medmicro/home.htm
_______________________________________________________________

"If we knew what it was we were doing,
 it would not be called research, would it?".   --   A.Einstein
_______________________________________________________________



More information about the Methods mailing list