Ligation into a BAC? - (Jul/31/2012 )

Hello all,
Im new here, so sorry if this is asked repeatedly, I didnt see anything in my search.
Im trying to ligate some fragments into a BAC 15.9kb and am running into some problems. I extract the plasmid dna using an alkaline lysis method, yielding hundreds of micrograms from a 150 ml culture, suspending in TE buffer. I would like to add two fragments into single sites on the plasmid, one 3.8 kb and the other a 90bp mcs I have created. The larger fragment is to go first, into a PacI site that has been added by PCR to the fragment, and is native to the BAC. I digest each following neb protocols, digesting 1ug in a 50ul reaction. This is the first problem, taking 10ul of each reaction shows very little, if anything, on a gel. I would assume that the concentration should be 20ng/ul after digestion, which i should be able to see. The bac is treated with antarctic phosphatase for 15 min and killednin 65 for 5 min. I use 20ng vector and have tried 1:3 and 1:8 molar ratios. The mix is added to t4 for 1 hr at rt then 1ul is added to 20ul of dh10b cells and electroporated. This shakes at 37 for one hour before 50 ul is plated overnight. Ive never gotten any colonies, though i also have not run the undigested control unfortunately, our cell stock is running low right now. Im wondering if anyone has any thoughts or tips? Thanks for the help!

I think the amount of DNA you are using is probably too low. Because the plasmid is at least 5 times bigger than those used in normal cloning 3-4kb you will need to add 5 times more DNA to get the same number of physical copies in your reaction. The cloning will also be much less eficient because the bacteria normally preferentially take up smaller DNA fragments. Your vector is pretty big.

I would be digesting at least 5ug of DNA for the reaction and add a lot more into the ligation and transformation. Also try the ligation overnight at 16 degrees, that might help but only a bit. I think doing the uncut DNA control would also be good to check the selection, plates electroporation etc. Good luck!

If you are getting no colonies, it is likely that your competent cells are not competent enough. You should quantify their competence, by transforming serial dilutions of a known plasmid (ideally the plasmid you are using). You should be getting at least 10^8 CFU/ug transformants. Less will give you problems, of the kind you are seeing.

Your inability to see DNA is troubling. Have you run a lane with the DNA only? Perhaps you have much less than you think (or none). RNA contamination can fool most DNA quantitation techniques.