Protocol Online logo
Top : Forum Archives: : Molecular Biology

KpnI Ligation Mystery - Bidirectional Cloning Working Unidirectionally (Jan/19/2006 )

In 10 years of molecular biology I have never seen this. I have a fragment with KpnI site at both ends. I am trying to insert this fragment into PGL3-basic luciferase vector. I have done 84 minipreps now, and all the positive clones I've gotten are in one direction (my luck, this is the wrong direction). My only explanation is that there's some sort of steric effect selecting for this direction of cloning. But, I've never seen this before and any thoughts would be appreciated.

Thank you,
Nokhodi blink.gif


Is it a gene that you are cloning? It could be toxic if expressed and perhaps in the orientation that you want, the gene is being expressed from a promoter behind it. For example, I have found that when cloning genes into the lacZ gene of pBluescript, I can get some expression of the gene from the lac promoter. In your case, if the gene is toxic, any clones that express the gene will not grow. I wonder if you could run a PCR on the ligation reaction using a primer from the insert and a primer from the vector to see if you are getting plasmids with the insert in the orientation you want. That would at least tell you that the ligation is working...


good point. but the fragment is actually part of a promoter--noncoding sequence. but maybe the DNA is actually being substrate for some sort of modification which affects the vicinity of the insert and the Amp resistance gene. this is all very far fetched but, i have never had so many one directional clones.


how are you testing for the correct directionality?


I use two different restriction sites thus only one direction is possible in the clone. Have u given a thought to trying that?


Why not use two restriction sites?

It's not any more difficult or expensive than single restriction site cloning.