Ligation into a BAC?
Posted 31 July 2012 - 05:08 PM
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!
- Gnatnog likes this
Posted 06 August 2012 - 11:28 AM
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!
Posted 07 August 2012 - 03:54 AM
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.