I-SceI meganuclease - restriction trouble of BAC DNA (Jul/03/2005 )
Has anyone used meganuclease I-SceI to linearize BAC?
We are using it but have not got good results.
The digested DNA was ProK/Phenol/ChCl3 treated and then analyzed on PFGE. We could see only a smear on the gel. The DNA seems to be good quality as the NotI digestion gave us very clear single band of the insert. The I-SceI binds to DNA non-specifically, and the I-SceI treated DNA needs to add SDS before running gel. I tried this protocol but got same smeared gel pattern.
Is anyone know how to use the nuclease?
BACs are typically long DNA fragments -- treating them like plasmids will result in smears. The preparation of long DNA, and its cutting, is typically done in samples which have been lysed and purified in agarose, and the digestion is then done in agarose, as well, and loaded into the PFGE apparatus as a slice of agarose. If you are phenol/chloroform purifying your DNA, you are almost certainly shearing the DNA during this process, leading to smears. Read up on protocols for handling long DNA fragments.