I have a problem when trying to clone a 1kbp gene into a vector. The vector is about 8kb. I PCRed the gene, which works beautifully. Then I digested both the insert and vector using XhoI and BamHI. The DNA was gel purified. Everything seems OK.
However, after I ran the ligation and used the ligation mixture to transform E. coli cells, I did not get a single colony! I did my ligation using Roche rapid ligation kit. Since I did not get colonies, I went back to repeat the ligation experiment and checked the ligation mixture on a gel. I also did a control experiment with both insert and vector, as well as the ligation buffer, but omit the ligase. On the gel, I found the control mixture contains both the linear vector and insert, as I expected. For the mixture with ligase, I saw a smeared band with high molecular weight (higher than the linear vector) and both the bands corresponding to the linear vector and insert disappeared. Therefore, I concluded that at least the ligase works and both the insert and vector can be ligated into something, though I cannot say they can be ligated into the product I want. However, I still have no colonies, even use ligation mixture that I clearly proved to contain some products with above method. I also set a ligation reaction with only the vector. Normally, two vectors should be ligated together and this should give you some colonies (though these colonies are useless for cloning purpose). However, I still have no colonies. Now I start to suspect the competency of my cells(DH5alpha) and even change competent cells to Agilent XL-2 ultracomp cells and, I also did a positve control using intact vector during the transformation, which leads to lots of colonies. Therefore, the cells seem to be OK.
Right now, I do not have any more ideas on how it should work. Do you have more ideas how to trouble shoot this problem?
Still no colonies……with no results………..