Newbie to smaI blunt end cloning! - (Jul/11/2013 )
I am a newbie to Blunt end cloning with smaI. I am trying to clone a pfu PCR product into pBSKS vector at SmaI site. Can I just cut the vector with smaI and (not the amplicon) ligate it with the amplicon? I dont want to cut the amplicon as I dont know if it has a smaI site. Do I need any polishing step for vector such as phosphatase treatment or so.??'
Religation of the smaI cut vector will be a problem unless you dephosphorylate it. Do this with a heat sensitive enzyme, and use the least amount for as short a time as possible. You can test this by ligating and transforming the vector-only DNA, and checking the amount of background. Use "quick ligase" buffer for this blunt ligation. You will also need to either order 5' phosphorylated oligos for your PCR, or phosphorylate the insert with PNK. This would not be a favorite way for me to do anything. Have you thought about alternatives?