Incomplete genomic digestion for Southern - (Dec/21/2005 )

Hey Everyone,

I am trying to screen founder mice for a transgenic mouse model. I have purified the DNA and performed a AscI enzyme digestion on the samples. I used 3ul (10,000U/ml) of the enzyme in a 40 ul reaction containing approx 5ug DNA. O/N incubation @ 37oC. Upon running the gel I found only a smear in approx 25% of the lanes whereas the other lanes looked to have incomplete or no digestion at all.

I made a mastermix of the enzyme and also the DNA preps were all prepared at the same time using the same protocol. This is my second transgenic project and the previous one which used HindIII worked well everytime.

Does anyone have an idea of what could possibly be causing the inconsistency. I am quite limited to what enzyme to use for a single cut within the transgenic cassette (AscI or SalI)

Kind regards

Hey, Happy new year
I am afraid you met the CpG problem. AscI cleavage of mammalian genomic DNA is blocked by CpG methylation. So is SalI. I never tried AscI, but had some experience on SalI, it works sometime, but not always. Do you have the other choice?
H