Gene insertion without desired unique restriction sites - (Sep/27/2009 )

Hello friends,

while working on a cloning experiment i came across a problem.
I have to clone a gene into a binary vector that has been modified and contains only two restriction sites-NcoI and XbaI.
I need to use only this one to clone some genes that have either of the two or both the sites internally.
How can i use the same vector,ligate my insert into it and prevent my gene from being chopped off on restriction digestion with the above enzymes?

SOUMYA RAO on Sep 27 2009, 02:32 PM said:

Break out your New England Biolabs catalog and look up compatible cohesive ends. For example, I know SpeI and NheI (both common enzymes most people have) are compatible with XbaI -- so if your gene doesn't contain these sites, then those could be used. The compatible ends for NcoI are less common enzymes, but then again, you could use any enzyme and blunt this end, and blunt the NcoI site; with a single cohesive end, the cloning is still pretty easy. Warren..

Should you be unable to use restriction enzymes that have compatible cohesive ends (ei NheI, SpeI), you could consider changing the restriction site in your binary vector to something more suitable. You can

1- clone in a synthetic oligo linker into your vector. This linker can be designed to delete the restriction site that it goes into.

2- you can PCR amplify your vector with primers that contain your desired restriction sites.

perneseblue on Sep 28 2009, 02:32 PM said:

Thanks a lot,
I will surely consider this if don't get any compatible sites for NcoI.
Soumya.