cloning a pfu amplified fragment in T vector
cjmcdermottroe from gmail.com
(by cjmcdermottroe from googlemail.com)
Sat Feb 6 19:27:33 EST 2010
Please tell me if i'm wrong but i always assumed that kod polymerase doesnt touch the restriction tags because the unpriming region of the primer is distal to the site of polymerase activity (when priming against template). In the case of extending when the newly generated strand is used, the pol is none the wiser and thus will quite happily generate a mutated sequence (since it arises from a mutated sequence).
Subject: Re: cloning a pfu amplified fragment in T vector
From: "Cathal Garvey" <cathalgarvey from gmail.com>
Date: 06/02/2010 10:40 pm
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...
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 <rahimpour_a from yahoo.com> wrote:
> 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?
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