reamplification mtDNA

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Tue Jul 12 10:19:12 EST 1994


Hi Brian
We have been amplifying a gene from a lambda gt10 library and found that 
no product was visible after 30 cycles. If we took 5ul of this reaction
(reaction volume was 50ul) and reamplified for a further 30 cycles we got 
a nice clean product that could be cloned and sequenced.  The one problem
we did come across however is that the errors introduced by Taq increase
with the number of cycles. With proof-reading polymerases the problem may 
not be so bad, also we were amplifying a 1.4kB fragment.  I think with a
smaller fragment errors are less likely to occur.
Hope this is of some help
Good luck
Emma



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