cloning to pET28a (View forum version)



osnattirosh

Posted 13 August 2012 - 03:46 AM

i've been trying to clone a gene encoding a membrane protein fused to GFP into pET28a and cannot get colonies in transformation to Top10 or C43(DE3) chemically compatent cells, or the colonies i get do not have the insert of my gene of interest, any suggestion?

ascacioc

Posted 13 August 2012 - 04:47 AM

we need more information... Tell us what you did.

It seems that you have a religation problem in the few colonies you get: try to digest the ligation reaction with a 3rd restriction enzyme that is in the middle of MCS in between the 2 restriction enzymes that you have used for cloning. Like this you linearize the relagations (the only one containing an intact MCS). But I believe that this will not solve your problem but rather make you get rid of the few clones you have.

Andreea