PNK treating insert? - (Mar/30/2007 )
Hi all,
This is my first question on this site so I am curious to see if it helps!
I am doing a simple blunt ligation of insert and vector and plan on dephosphorylating the vector and using PNK on my insert.
Apparently you can do the PNK treatment and ligation both in the same buffer (T4 DNA ligase buffer). However, if I don't heat inactivate the PNK I am assuming my vector will get re-phosphorylated and self ligate. If I do heat inactivate will I destroy the ATP requried for the ligation reaction?
Am I just being incredibly dense??
This is my first question on this site so I am curious to see if it helps!
I am doing a simple blunt ligation of insert and vector and plan on dephosphorylating the vector and using PNK on my insert.
Apparently you can do the PNK treatment and ligation both in the same buffer (T4 DNA ligase buffer). However, if I don't heat inactivate the PNK I am assuming my vector will get re-phosphorylated and self ligate. If I do heat inactivate will I destroy the ATP requried for the ligation reaction?
Am I just being incredibly dense??
Hi,
I'm also dealing with blunt ligation and I was told, that it's necessary to do a PCI extraction after PNK, overwise (you're assuming right) will get re-phosphorylated and self ligate.
Hi
Ihope this helps u.. u can give ur insert PB wash (qiagen kit) to purify and use it for ligation..ul have to add fresh buffer for ligation . Alternatively u can phosphorylate ur primers by pnk treatment and dirctly use ur pcr product , after gel elution, to set up a ligation. (phosphorylation of smaller fragment is more efficient..)
gud luck