2 replies to this topic

[TracyDuke]

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.

[klinmed]

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.

[TracyDuke]

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.