ligation problem by using single RE - (Nov/18/2008 )

Hey, recently I am trying to insert a ~ 1.2kb fragment into a 7.4 kb vector by using a single RE (xbaI). I cloned the PCR product (both end has xbaI linker) into a TOPO zero blunt vector, and digested with xbaI, gel purified the fragment. Vector is also cut with xbaI and CIP treated, then gel purified. Then I tried different ratio like 1:3, 1:4, 1:5, .. even upto 1:10, but no colonies on the plates. I tried different ratio, different batch of plates, different competent cell... No matter what I tried, I didn't get one colony at all. What should I do? I would appreciate if you have any suggestions. Thanks

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You are purifying the ligation with gel extraction???? write what you do step by step because I'm don't understand what you mean with this: "I cloned the PCR product (both end has xbaI linker) into a TOPO zero blunt vector, and digested with xbaI, gel purified the fragment. Vector is also cut with xbaI and CIP treated, then gel purified."
What do you digested and what you purify with gel.

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Have you tried doing a test with just your vector- not CIP treated, to see if it will religate, and have you tested your ligase to see if it's working?

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Sorry to make you comfused. I cloned the sequence of interest into a TOPO vector, and sent for sequencing. After confirmation, the plasmid was digested with xbaI (the sequence has xbaI linker on both ends) for two hours. Then I ran a gel, cut the right band, and did the gel purification. The vector that I want to insert the sequence into was digested with xbaI and CIP treated (incubated at 37 C for 30 min, inactived at 65C for 20 min) Again, I ran a gel and gel purified the digested vector. I tried differet ratio of insert: vector, tried different ligase, and different competent cell, no colonies at all. The non-CIP treated vector only control confirmed that the digested vector religated. I hope my explanation can make you clear now. Thanks for any suggestions.

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Yes, I tried this. The non-CIP treated vector only control plate has lots of colonies that confirmed it is religated. And I know the ligase it is working. Thanks

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Perhaps you're not getting completely rid of the CIP during the cleanup process. A CIP treated vector with no insert should give you at least a few colonies (according to NEB, about 3-5% of non-CIP treated cut and religated vector control). Perhaps try to scale back on the amount of CIP you are using??

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I agree with smu2. Try to add less CIP.

You may also get an improper buffer for ligation, if you precipitate your DNA with sodium acetate, or still bring the CIP down with the precipitation. I suggest you try to gel-purify your vector and insert with commercial kits, such as QiaExII, or another gel purification kit. Its gives out pure DNA and gets rid of all the salts that would otherwise compromise the ligation reaction.

Hope this helps!

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The problem could be that you are not having a good yield of DNA because of gel purification. Try to use a column purification. If the vector is religated there is a possibilty that the insert is loosing the terminals that the vector recognize (another problem do to gel purification).

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Thanks guys. I always do gel purification with Qiagen kit. But I will try to use less CIP. Loss of sticky ends of insert is also my thought, but I don't know how to avoid it. I was gentle enough to handle all of the reactions, no votex at all. Anything else?

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