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question about blunt ligating Taq-amplified PCR product - (Apr/23/2010 )

I have PCR product amplified by Taq polymerase. I am trying to blunt ligate the PCR product into a blunt-cut, dephosphorylated vector. Since Taq creates 3'-A-overhang, I used T4 DNA polymerase to blunt the ends of the PCR product.

My question is: After T4 DNA polymerase removes the 3'-A-overhang, are there phosphate left on the ends of the PCR product? If not what do you use to phosphorylate the DNA ends? Thanks.

-TracyDuke-

TracyDuke on Apr 23 2010, 09:29 PM said:

I have PCR product amplified by Taq polymerase. I am trying to blunt ligate the PCR product into a blunt-cut, dephosphorylated vector. Since Taq creates 3'-A-overhang, I used T4 DNA polymerase to blunt the ends of the PCR product.

My question is: After T4 DNA polymerase removes the 3'-A-overhang, are there phosphate left on the ends of the PCR product? If not what do you use to phosphorylate the DNA ends? Thanks.

Blunting with T4 poly will leave 3-OH. Ligation requires 5-phosphates and you must add these with T4 polynucleotide kinase + ATP. The protocol can be found in most enzyme catalogues.

Hope this helps.

-klinmed-

klinmed on Apr 23 2010, 02:01 PM said:

TracyDuke on Apr 23 2010, 09:29 PM said:

I have PCR product amplified by Taq polymerase. I am trying to blunt ligate the PCR product into a blunt-cut, dephosphorylated vector. Since Taq creates 3'-A-overhang, I used T4 DNA polymerase to blunt the ends of the PCR product.

My question is: After T4 DNA polymerase removes the 3'-A-overhang, are there phosphate left on the ends of the PCR product? If not what do you use to phosphorylate the DNA ends? Thanks.

Blunting with T4 poly will leave 3-OH. Ligation requires 5-phosphates and you must add these with T4 polynucleotide kinase + ATP. The protocol can be found in most enzyme catalogues.

Hope this helps.


Thank you very much Klinmed.

-TracyDuke-