How to fill in 5` overhangs after Restriction digestion - (Mar/15/2006 )

Hi there,

I tried to ligate my digested PCR product (BamHI and EcoRI) into pCDNA3.1+. Unfortunately, I didn`t get any positive clones. Now I have some of the digested PCR product left. How can I fill in the 5` overhangs? Will a PCR generate the complete sequence? I would like to get blunt ends somehow to ligate the insert into pGEMT-easy vector (after A-tailing).

Thanks for helping,

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from NEB:

"Q10: Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?

A10: DNA Polymerase I, Large (Klenow) Fragment and T4 DNA Polymerase (NEB# M0203) are the best choices for this application. DNA Polymerase I, Large (Klenow) Fragment can be used at 25°C or room temperature, but T4 DNA Polymerase must be used at 12°C due to its robust exonuclease. Both work well in a wide variety of buffers. Vent DNA Polymerase (NEB# M0254) and Deep Vent DNA Polymerase (NEB# M0258) can also be used but ThermoPol buffer must be used, the reaction temperature is high, and the enzyme cannot be heat inactivated. Mung Bean Nuclease (NEB# M0250) will chew back 3' overhangs but the strong exonuclease activity combined with the lack of polymerase activity yield a lower percentage of blunt ends.
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Yes, a single PCR extension will do it. Incubate at 72oC for 5-10min in 1x Taq buffer with standard Taq (0.2U) and dNTPs (~80uM working). This will fill the 5' overhangs and add 3'-A tails in one step. Purify the products on a spin-column immediately after the incubation, and ligate the DNA immediately after purifying.

As aimikins posted, Klenow, Vent or Deep Vent will also work, but you'll get blunt ends which still need 3'-A tailing.

good luck.

Del.

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