9 replies to this topic

[sara.r]

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

[Eve]

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.

[bob1]

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.

[sara.r]

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

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.

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?

[gogreen]

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.stratagen...ct.aspx?pid=546

gg

Edited by gogreen, 29 January 2010 - 11:35 AM.

[klinmed]

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

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

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.

[hanming86]

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

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

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.

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?

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.

[Biocat]

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

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

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

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.

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!

[bob1]

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

[molstudent]

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

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

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

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.

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?

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.

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."