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cloning a PCR product. Taq pol or High Fidelity pol? - (May/20/2010 )

Dear all hi!

Im trying to clone a pcr fragment (900bp) in my pegfp-n1 vector.

I was using FastStart High Fidelity PCR System from roche and my flanking end primers.
I did everything by the book and when I did the ligation i was getting an empty plate.
I tried everything I changed everything and nothing!!

Then I changed the polymerase to a simple Taq form takara.
AND the ligation worked!!!

I believe that the exonuclease activity of the highfidelity mixture degraded my flanking ends of my primers.

Does anyone else has the same experience?? or the same opinion?
Is there a way to bypass this problem and use a high fidelity polymerase enzyme?
Or is anybody using a high fidelity enzyme for cloning restricted PCR products?

Thank you very much in advance.

t

-tasosp-

If you are doing digestion after PCR then simply clean-up PCR reaction (spin column) to remove polymerase before adding restriction endonucleases and RE buffer.
I always do spin column clean-up between steps like PCR - blunting - digestion - phoshorylation - ligation etc. even when it is not necessary according to some protocols. You can also omit heat-inactivation in each step. You can recycle same spin column for these multiple steps.

-vladooo-

tasosp on May 20 2010, 04:34 PM said:

Dear all hi!

Im trying to clone a pcr fragment (900bp) in my pegfp-n1 vector.

I was using FastStart High Fidelity PCR System from roche and my flanking end primers.
I did everything by the book and when I did the ligation i was getting an empty plate.
I tried everything I changed everything and nothing!!

Then I changed the polymerase to a simple Taq form takara.
AND the ligation worked!!!

I believe that the exonuclease activity of the highfidelity mixture degraded my flanking ends of my primers.

Does anyone else has the same experience?? or the same opinion?
Is there a way to bypass this problem and use a high fidelity polymerase enzyme?
Or is anybody using a high fidelity enzyme for cloning restricted PCR products?

Thank you very much in advance.

Primer degradation is quite often seen with proof-reading polymerases. Some enzymes are worse than others. I have found pfu to be particularly troublesome.

One way to minimize the effect is to limit the annealing and extension times as much as possible. A much simpler way is to use two phosphorothionate linkages between the last 3 bases. This makes the primers resistant to degradation by the polymerases. This modification is done by most companies and is cheap.

Hope this helps.

-klinmed-

Klinmed thanks. Sounds that you know what im talking about....
Can you please explain to me in more detail how my primers will be? Ive got a 30 base pair primer. The 20 3end nts are gene specific. My 10 5end nts have the restriction site. Which part should I modify? Please if possible attach a website that I can check.

Thank you very much!

t

-tasosp-

tasosp on May 21 2010, 07:27 PM said:

Klinmed thanks. Sounds that you know what im talking about....
Can you please explain to me in more detail how my primers will be? Ive got a 30 base pair primer. The 20 3end nts are gene specific. My 10 5end nts have the restriction site. Which part should I modify? Please if possible attach a website that I can check.

Thank you very much!

t


See the "classic" paper:

C M de Noronha and J I Mullins, Genome Res. 1992 2: 131-136.

This should answer most of you questions.

-klinmed-

If you got PCR product from hi-fi polymerase reaction then you DO NOT have a problem with primer degradation DURING PCR.

-vladooo-

vladooo on May 22 2010, 09:23 AM said:

If you got PCR product from hi-fi polymerase reaction then you DO NOT have a problem with primer degradation DURING PCR.



Ive got pcr product but not ligation, after restriction.
The degradation happens to my flanking ends of the primers. Not to the whole primer. Thats what i beleive. and I think that is what is going on.
I think the answer is the modification of the primers as klinmed proposed. Im looking in to right now.

Do you get colonies after using HIFI polymerase, and restriction reaction (not TA cloning)?

Thanks guys!!

-tasosp-