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.
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.
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-