EcoRI cloning - correct orientation - Preferential orientation? (Apr/04/2005 )
Hello all,
I am cloning an EcoRI fragment (cut out of pCR-Topo II) into EcoRI-cut pBluescript SK- and I need it in a specific orientation. The ligation reaction went well, but now I'm screening clones for my desired orientation and have found them all to be in the undesired orientation (23 out of 23 screened). I thought it should be a 50% chance of getting my insert in the right direction. Is there some reason this would be cloning preferentially in one direction? Or am I just getting unlucky and need to screen more clones?
I would say that what you are experiencing is just another example of Murphy's Law at work. I can not see any reason why one orientation should be preferred over the other one when cloning into bluescript. Just keep on trying. Good luck!
I am cloning an EcoRI fragment (cut out of pCR-Topo II) into EcoRI-cut pBluescript SK- and I need it in a specific orientation. The ligation reaction went well, but now I'm screening clones for my desired orientation and have found them all to be in the undesired orientation (23 out of 23 screened). I thought it should be a 50% chance of getting my insert in the right direction. Is there some reason this would be cloning preferentially in one direction? Or am I just getting unlucky and need to screen more clones?
I kind of thought so, but thanks for the confirmation.
Back to the ol' drawing board
B)