Posted 12 February 2010 - 04:51 PM
Please help me out,
Posted 12 February 2010 - 05:08 PM
Posted 16 February 2010 - 10:47 AM
So, are you doing a PCR with phosphorylated primers followed by blunt ligation? What enzyme are you using for th PCR?
yes and I'm using Pfx polymerase from invitrogen
Posted 16 February 2010 - 02:49 PM
Posted 17 February 2010 - 12:28 PM
How are you preparing your vector? Can you do a control blunt ligation of the vector only and get transformants?
The vector was already made and was given to me. It has got the backbone of pBSK vector. All I am doing is creating a mutated site via mutant primers. The primers abut each other with one going in the sense direction and the other going in the antisense direction. Once the primers have been completely amplified, the ends of the primers which contain phosphate groups are ligated using the blunt end ligation protocol. My positive control seems to give me colonies after I transform it.
I have another question: If I digest my control plasmid and ligate it again, would I be able to see a supercoil band if completely ligated, or is supercoil generally not produced upon ligating a digested product.
Posted 17 February 2010 - 04:23 PM