> Dear all,
>> We got a problem fpr PCR-cloning a gene. The primers we are using are very
> long(~60 bases), but for each primer, only 29 bases are complementary to the
> template(we have to use such long unmatched extensions). I wonder how to
> caculate Tm. On the other hand, I'm not sure if I can use a standard method
> (ie, 94C, 45s; Tm-5C, 30-60s; 72C, 60s; 25-30 cycles).
>> Your help will greatly appreciated.
>> Rick(change 'll' to '11')
I am also amplifying sequences using tagged primers and my advise would be to
take Tm of the matching sequences to optimize your amplification protocol! I
usually use standard buffer and up too 800bp 40"/95°-20"/(Tm-2°)-40"/72° for 33
cycles (1U Taq/50µl) is sufficient ( I would also recommend to mix 1/20 unit
of Deep Vent Polymerase to your NON-proofreading Polymerase-!) further
optimization if there is no product : add 4% DMSO to your RXN or 30mM TMAC
(Tetramethylaammonium.Cl, use 1.5M stock) or combine both enhancers!
(send me your results - would give you further assistance)
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