help on directional cloning - (Jun/25/2008 )

Hello, everyone!
I have a problem with my directional cloning. I try to clone one insert (PCR product) which is 1.5kb into a vector which is 8.9kb.
To do this, I first cut my vector with FseI and NheI restriction enzymes. By this I got two fragment, one is 5.5 kb, the other is 3.4kb. After that I gel purified the double digest vector to get 5.5 kb fragment for the ligation step. For my PCR product, I also double digest it with FseI and NheI after digest I purified the PCR product with GE PCR purification Kit. Next step, I combined the 5.5 kb fragment and PCR product together and use T4 DNA ligase to perform the ligation, then transformation into DH5a and spread on selective plate.
Finally, I did the Miniprep and double digest with other two enzymes to check my insert. I got some undesired results. Some of colonies don’t have insert and by looking at double digestion results, it showed that the 5.5 kb fragment self ligated which is impossible because those two cut sites are NOT compatible.
Did someone can help me for this question???

Thank you very much.

Could anyone give me some idea, please!!!

Self ligation is not impossible when a vector is cut with two non-compatible restriction enzymes. There are two situtations where you can get vector self ligation. Some vectors will only be cut once due to incomplete digestion and therefore easily self ligate if you don't dephosphorylate the vector. However, the difference in the size of completely and incompletely cut vector is quite large for you so this is not the problem. What could be the problem though is two vectors ligating to each other if the vector is not dephosphorylated. This is a possibility. Moral of the story: even if you are using two non-compatible restriction sites, dephosphorylate the vector.

I think that most people don't do a detailed enough digest to screen their clones. At a minimum, digest the clones in the insert and on both sides of the vector to get a better picture of what is going on. A couple of different digests gives you more information about the identity of the clones if one digest gives a strange result.

Also importantly, try to work out exactly what the "undesired" clones are. This will give you the biggest clue as to what is going on. Working out what strange digestion patterns are caused by is often the biggest clue and you often identify a simple error in your procedure by this method.