ta cloning of pfu amplified fragment - (Jan/28/2010 )

hi
I want to clone a pfu amplified fragment in T vector but I dont know if it is necessary to purify it before adding A overhangs or I can simply use pcr product for next step, In the latter case is there any probablity that pfu remove a overhangs by its exonuclease activity?

thanks in advance

I would purify it first, but it was my understanding that PCR for TOPO cloning is performed with Taq which will add A overhangs on it's own with a high probability.

Next time PCR with Taq and you can eliminate a step in your process.

Continue to amplify with your proof reading polymerase. You should purify the product before adding the A overhangs as the pfu will remove them unless you go immediately onto the cloning step.

bob1 on Jan 28 2010, 04:10 PM said:

thanks for reply but how come in long pcr or high fidelity enzyme mixes that are mixture of tag and a proofreading enzyme we can use pcr product directly in t/a cloning? isnt there a risk of a overhang removal?

hi sara,

just saw this enzyme from stratagene...an enzyme like this might be of help...its a bit costly, but counting the cost of normal taq, a cleanup another enzyme for adelylation and the time taken, it might be helpful ! I am going to try one sooner or later.
http://www.stratagene.com/products/showProduct.aspx?pid=546

gg

sara.r on Jan 28 2010, 03:34 PM said:

Amplify your fragment with a proof-reading polymerase (always a good idea). Purify using a kit (eg Qiagen PCR), then incubate at 72 oC with dNTP + taq for 20 mins. The extendase activity of taq will add the "As". Purify and ligate as normal.

Hope this helps.

sara.r on Jan 29 2010, 05:42 AM said:

Well, in long PCR mix, the amount of proof reading polymerase is present in quite small amount. even if it removes it , the dominant Taq will add it back and there might be those without tail but perhaps at very minute amount.

klinmed on Jan 30 2010, 12:28 AM said:

But for my case, i failed to amply my gene with proof-reading polymerase (Roche high fidelity PCR master and pfu), so i just can amply gene with Taq enzyme, do you think i still can use optimize parameters to amply my gene with proof reading polymearse?
Many thanx in advance!

Both of the polymerases you mention should work if Taq does - what are your amplification conditions?

hanming86 on Jan 30 2010, 04:57 AM said:

This is true, from the invitrogen topo ta manual it says

"If you wish to use a mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess of a 10:1 ratio to ensure the presence of 3 ́ A-overhangs on the PCR product."