There are a few reasons why you can't get your band using the alternate
A purely theoretical reason:
Depending on the composition of your sequence, a high GC template may be
more difficult with an error-checking enzyme, perhaps because the template
cannot "mutate" to a more easily amplified form. For example, a knot of
high-GC DNA might mutate quickly using Taq, where there is no error
checking, because there is a "selective pressure" for template that can be
amplified properly. With an error checking enzyme, this is nigh impossible.
Leaving that purely theoretical reason aside, it's more likely that pfu's
different amplification rate and reaction preferences are to blame. As it is
a different enzyme, it can't be assumed it favours the same Mg
Seeing as you've already tried changing all of these things, it could simply
be that the extension times are too short or long; taq and pfu amplify at
very different rates.
Finally, having tried to amplify a horribly complex sequence with an error
checking enzyme myself, and having little success worthy of cloning, I would
reccomend that you consider having the DNA amplified if you need it only
once. If you go over to MrGene.com, they can synthesise the sequence from
scratch and send it to you in an ampicillin resistant vector at a
competitive price! Just don't trust them to do it as quickly as they
advertise if it is a high GC sequence..
2009/8/10 malihe keramati <[Only registered users see links. ]>
Kiva.org - Loans That Change Lives