BAC isolation kits and differences in nanodrop readings - (Nov/16/2011 )
We isolate BAC DNA in my lab. We have a number of kits available for the isolations. The kit that we use the most (AquaPlasmid) is quite expensive and difficult to get consistent results with (in fact, I am the only one in the lab that seems to have the "touch" and can get any DNA with it). It would be nice to use a simpler kit, but when we do, we get very strange reading from the Nanodrop and the DNA does not seem to be of very high quality. Our typical yield with Aquaplasmid is 50-100 ng/ul, as read by the Nanodrop, and the DNA bands look very strong and the DNA nick translates well. With other protocols and kits, the nanodrop reads 2000-3000 ng/ul but there is not really that much DNA (10-20ng/ul as measured with picogreen) and the DNA bands look faint and do not nick translate well. Does anyone know what kind of contamination that the nanodrop could be reading and what could be causing it? It is not bacterial DNA or RNA.
The components of typical binding reagents such as guanidinium HCl strongly absorb at 230 nm, with bleed over to 260. Usually this contamination results from inadequate washing of a column. Some columns have a lip or ledge that can fail to be washed. Also, the outside of columns are often carriers of contamination into eluted DNA. We routinely wash columns twice, and then spin a third time to dry.