Blunt end cloning question - (Dec/19/2007 )
Hello. I`ve always cloned using the easy route of adding restriction sites that match the vector I`m cloning into, PCR, cut and paste. Unfortunately I `ve been struggling with my newest construct. My PCR isn`t working too well so I`m thinking about cutting my construct out of it`s current vector and pasting it into the new vector. I have to use the sites KpnI/NotI in the new vector. My current vector has a KpnI site I can use, but no NotI site. There is however a SmaI site I could use.
My question is this: Could I digest my new vector NotI, treat with Klenow, then digest KpnI, and finally ligate in my construct digested with KpnI/SmaI? SmaI is a blunt end enzyme and Klenow would convert NotI into a blunt end, but is it that easy or do I need to do anything else?
Thank you in advance for the advice.
In theory, your strategy should work. Could you use any other blunt end enzyme. My worry is about SmaI. Sma I is a blunt end cutter and it doesnt usually cut very efficiently, atleast for me.
i would suggest to go ahead with the plan and hopefully it will work.