Biotaq problems, please help!!

Oliver S. DePeyer saspeyer at reading.ac.uk
Thu Jan 9 07:01:07 EST 1997


I have been having severe problems with clones PCR'd using 'Biotaq' (made
by Bioline UK ltd). I have been suffering a very high number of point
mutations, i.e 3,4,5,6... or more mutations in a gene (MIP26) of C.800bp.
These mutations have been seen in 22 sequences which were derived from
PCRs done on different dates using different templates and primers, etc.
showing that this is a chronic problem, rather than a one-off.
Insufficient annealing temperatures is unlikely to be the cause since the
annealing temperatures were optimised as per RYCHLIK, SPENCER, RHOADS 1990
and were in the range of 55-58'C; previous researchers here in the Wolfson
Lab have got clones of perfect fidelity for MIP26 with temperatures as low
as 50'C! 

Since I understand that Biotaq is in widespread use, I would therefore be
very grateful if people could send me any protocols, advices etc. etc.
that they have used to get high-fidelity clones from PCR using Biotaq - I
was thinking that perhaps there is some subtle idiosyncracy in the
protocol as applied to this taq, such as a preference for atypical cycle
times or MgCl concentrations. 

If I was to use another taq instead of Biotaq, I would need one that gave
me A overhangs like Biotaq does, since all my primers are set up for
ligation into pGEM-T, which has complementary T overhangs. 

So, does anybody know of a more reliable taq that ALSO gives A overhangs?

Or, alternatively, does anybody know how I could get A overhangs out of
Bio-X-Act or PFU taq? These high-fidelity taqs USUALLY give blunt-ended
PCR products, but perhaps if you had a very long extension cycle... or
something... 

If anybody has any ideas, please contact me!!
PS If you want to check out the context in which this work is taking
place, please check out our web site... I'd love to have some comments on
it from outside my university.


			  Oliver de Peyer
			  Wolfson Lab 136
			School of AMS (wing)
		       University of Reading
			    PO box 228
			   Whiteknights
			     Reading
			    Berkshire
			     RG6 6AJ
			       U.K

Tel. +44 (0)1734 875123  extn.7889
Fax  +44 (0)1734 316671
o.s.depeyer at reading.ac.uk
http://www.rdg.ac.uk/Wolfson













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