Ligation of large DNA fragments (40 kb + 10 kb) - Does anyone have any suggestions? (Oct/23/2005 )
For the past five months I have been struggling with a ligation reaction. I have been trying to replace a 10 kb fragment in a cosmid of ~50 kb by ligating a 40 kb fragment to a smaller (mutagenized) fragment of ~ 10 kb. I have tried dephosphorylating the larger fragment to prevent religation of the unmutagenized fragment with the 40 kb fragment and, alternatively, gel purifying both fragments (or only the fragment from the digest reaction producing the mutagenized 10 kb frgament) to ensure only these two fragments are represented in the ligation reaction. Thus far I have not been able to obtain a clone consisting of the 40 kb fragment and the mutagenized 10 kb fragment. Does anyone have any suggestions?
usually ligation tells to get 100ng of vector. But this is for a 5-10kb plasmid. So, when you're facing a 40kb vector, i would use 400ng of vector, and the appropriate ratio of insert... so about 300ng of insert...
The difficulty you face is it is very hard to get both inter molecular and intra molecular ligation to both work well with large DNA fragments. You might want to try using a two step approach where you do intermolecular ligation for a short time (15sec) in a high concentration of PEG8000 and then dilute the ligation reaction in 1x buffer to a concentration that encourages intra molecular ligation (below 1ng/µl).
Are you using electroporation? You might also want to use a strain that is optimised for large clones too (DH10B and derivatives)