plasmid DNA not digested - (Jul/17/2007 )
I currently test a plasmid vector carrying an siRNA for the correct restriction sites, before I use it. I performed several reactions with 5-10U enzyme/ 400ng DNA. Any idea why I get no bands after restriction, but only the full length DNA? I incubate at 37 for 2h.
methylation? Some restriction sites can be dam or dcm or CpG methylated, protecting the site from digestion. Check your cloning strain, to see what kind of methylation it does. also check if if the sites are methylation sensitive.
Alternatively perhaps your DNA is too dirty to cut. 400ng sounds like a fair bit of DNA, what is the volume that your are cutting your DNA in?
Where did you obtain this vector, just to make sure the sequence is 100% correct (that the restriction site is really there).
Have you tried cutting with another enzyme (preferably one that is not sensitive to methylation, or one of which you are sure that the site it should cut is not methylated).
you could shRNA containing sequences if grown in inappropriate cells. sequence the vector to confirm presence of shRNA.