In my work I only use KOD Hotstart Proofreading Polymerase. In principal, it should also cut off tag overhangs and prevent use of restriction site tags, but in practise it doesn't.
It only takes a few mistakes on the part of the exonuclease for there to be a population of full length copies containing the tags. Once these full length sequences appear, they dominate due to a better binding efficiency... I think! Suffice to say I've had useful results doing this with KOD, which shouldn't be very different from PFU. Give it a try and see how you do. Bear in mind that Taq sometimes can't fill in the last few nucleotides of a template, so if you do decide to add tags with taq, make sure you account for how many nucleotides your restriction enzymes need to bind effectively and include these in your tags *after* the restriction sites themselves, plus a few. Oh, and forgive me if I'm mistaken, but it might be best to add the tags with taq before amplifying with PFU: If you amplify with PFU for 30 cycles and then do 2 cycles with Taq, you'll have 30 cycles of wasted template and only 2 cycles of useful tagged template. If you do 2 cycles with Taq and Tags, followed by 30 cycles of PFU, you'll have far more useful tagged template at the end. I hope this works for you! On 6 February 2010 11:38, Azam Rahimpour <rahimpou...@yahoo.com> wrote: > Hi > I need to clone a pfu amplified fragment in t-vector, in the vector manual > it is said that pfu pcr product must be purified before a tailing by tag > because pfu can remove a overhangs by its proofreading activity, but in > commercial high fidelity enzyme mixes which include a proofreading enzyme > with tag we can use pcr products directly for t/a ligation, so why in the > latter case A overhangs are not removed? > > regards > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
