directional cloning problem - two-digested vector and insert - ligate - no colonies?? (Dec/03/2008 )

Dear all!!
I'm not the most experienced person in ligation-cloning one could imagine. So, I'm just really curious, what did I miss..

The problem is as follows.
I have two plasmids - both initially from Invitrogen - pCR2.1-"X" (where "X" - is the gene of my particular interest, for which I know the exact sequence and size) and pcDNA3.1/V5-His/LacZ. Both of the vectors are the former TA cloning vectors, in which the "X" gene or LacZ were inserted correspondingly.

What I need is to replace the LacZ in pcDNA3.1/V5-His/LacZ with my "X". As the mentioned plasmids have very common restriction sites in polylinkers around the inserted genes, I tried to use the digestion-ligation cloning protocol.

What I did: cut both vectors by BamHI and XbaI (it allows to cut just around the TA-cloning inserted gene), inactivated to the possible extent the enzymes (80°C, 20 min), gel-purified the products (extracted and purified the core-part from pcDNA3.1/V5-His/LacZ - i.e. the part without LacZ - 5451 bp, and the insertion part with "X" gene from pCR2.1 - 1793bp). Both plasmids cut perfectly, giving the products of predicted size.
Then I perform the ligation of two purified fragments, using different ligation conditions and different vector:insert ratios...

What I see on the gel, when I load the ligation mixture after the reaction: bands of vector and insert, and also one band (and its size is close to the desired vector size) or more bands of higher molecular weight (number of bands depends on the reaction conditions).
What I see on the plate after bacteria transformation - no colonies.

I've also tried to use another pair of enzymes for digestion - the digestion worked also, I purified the products, run the ligation, transformed the cells and got.. No colonies (:

I'm pretty sure about my cells, about the antibiotic and restriction efficiency, the ligase also seems to be active and it worked perfectly previously (for blunt ligations in Gateway conversion system).


So, the experienced ones!! Please, help to resolve the mystery.
I should say, that the question is not urgent, as I've obtained the vector of interest by PCR and TA cloning. But I just want to know for future, where was my mistake in the ligation cloning.
Thank you in advance!

Hi Ina, I got similar trouble some time ago. Even you are pretty sure about your protocol here are some BIOFORUM suggestions that helped me :

1. cut your gel as quickly as possible, do not UV-illuminate your DNA for too long.
2. lower the DNA amount in ligation, use let say 20ng of vector and appropriate amount of the insert.
3. add just as little as 2-5ul of your ligation to cells (less is sometimes more).
4. try to do all the steps (restriction, purification, ligation) in one day, do not freeze your DNA.

Good luck

Hi Ina, I got similar trouble some time ago. Even you are pretty sure about your protocol here are some BIOFORUM suggestions that helped me :

1. cut your gel as quickly as possible, do not UV-illuminate your DNA for too long.
2. lower the DNA amount in ligation, use let say 20ng of vector and appropriate amount of the insert.
3. add just as little as 2-5ul of your ligation to cells (less is sometimes more).
4. try to do all the steps (restriction, purification, ligation) in one day, do not freeze your DNA.

Good luck