cloning a pfu amplified fragment in T vector

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Sat Feb 6 17:20:19 EST 2010


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 <rahimpour_a from 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
>
>
>
>
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