I recently tried a low-cost protocol to isolate BAC DNA that I will use as substrate for multiple digests. The protocol is just alkaline lysis with RNAse in the resuspension buffer. After centrifuging the neutralization product, I did an isopropanol precipitation on the supernatant. I ran the resuspended pellet on a gel, it looks like there is probably a BAC present, but the image is overwhelmed by a very bright low molecular weight blob. Is this the rNTPs from from the RNAse activity? Is there a good way to get my BAC without this low molecular weight by-product? The gel image is attached.
Thanks!
my guess is that the RNAse treatment did not worked; your band is RNA. just add RNAse A 1ug/ml in your DNA resuspension buffer (Tris Edta or water)
@Didier Poncet - I will retry the RNAse treatment, but I wonder why all of the RNA would be smaller than 100bp? Also, would phenol/chloroform extraction and/or isopropanol precipitation remove the degraded RNA remains after RNAse treatment?
by adding RNAse A in your bacmid DNA prep you will not see RNA anymore or only a faint band; for restriction digest analysis (and for a lot of other uses) this is enough. if you need very clean DNA then another purification step after RNase treatment will be necessary; but in that case I would do another prep with a fresh RNase prep (but RNase A is a very stable enzyme)