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I am conducting a mutagenesis on a gene in vivo of which I need to ligate into an expression vector. The primers I have designed overlap restriction sites of which I plan to use to ligate into the expression vector. The parts between the primers I will try to create mutants with error prone PCR.

My concern is the 3' A overhangs that are created from Taq polymerase. How prevalent is this? If it is highly common, what is the best way to account for these additions when trying to ligate my PCR product into the vector?

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I am not sure of a case where Taq doesn't add 3' A overhangs.

You could use TA cloning to clone your PCR product. The basic principle is to use a vector with 3' T overhangs. If you have a vector without these overhangs, you can use a terminal transferase + dTTPs to create them (or even Taq, but this is not as great because it adds a purification step to isolate the vectors with overhangs; terminal transferase is much more efficient). You can then ligate your PCR product that has a 3' overhang.

Protocols for adding the 3' T overhangs to your vector are given in Zhou and Gomez-Sanchez 2000.

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  • $\begingroup$ T4 polymerase does not add overhangs (it adds them but then deletes them), but I think this polymerase has too high fidelity for mutagenesis. $\endgroup$
    – Kevin
    Apr 3, 2013 at 3:39
  • $\begingroup$ From what I understand, the A' overhang does eventually fall off. However, TA cloning is a much easier route if you have a TA vector. $\endgroup$
    – bobthejoe
    Apr 3, 2013 at 8:35
  • $\begingroup$ @Kevin: I have revised my answer to include a description of TA cloning, which does not involve T4 polymerase at all. (I assume you confused the letter A for the number 4.) $\endgroup$
    – blep
    Apr 3, 2013 at 22:05
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    $\begingroup$ In any case if restriction sites flank the PCR product, then you can go ahead and perform a restriction digestion and ligation. If you are too concerned by the A overhang then you can use Pfu polymerase for PCR or alternatively treat the PCR product with Klenow. $\endgroup$
    – WYSIWYG
    Apr 8, 2013 at 13:39
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Use pGemT to clone your PCR product into. It is exactly designed to take products with 3' overhangs and features blue/white selection so is pretty easy to do! Then you would cut out of pGem with the restriction sites you added during your PCR and ligate it into your expression vector.

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