Weird results for gDNA extraction... - (Feb/29/2012 )
Hi all,
I've been getting some very weird results from some gDNA extractions I have attempted recently. When I run my final product on a gel (see pics), I get that there is very little gDNA (first two wells after the ladder in the whole gel image; the other four bands are lambda DNA of known concentrations), and some weird bands that occur further down in the gel. When zoomed (zoom pic) in, there are three distinct bands. Could this be plasmid DNA or is it RNA? The sample was treated with RNAse, so there shouldn't be any RNA, but perhaps I am not using enough.
It depends quite a lot on what you are trying to extract the gDNA from. If it is bacteria (given that you suggest the smaller bands might be plasmid), then those bands could well be plasmid, though I would expect plasmids to run as 1-3 larger bands which depend on the size of the plasmid and the state of the plasmid at the time. These would look much like the two bands you are getting in the full gel picture, with the upper band on the top loadng dye front.
gDNA would usually stick close to, if not in the wells.
The smear at the bottom of the gel may be either digested gDNA or RNA.
I am trying to extract the gDNA from bacteria. If the smaller bands are not plasmids, what else could they be? If they (or the larger bands) are plasmids, at what step would I be losing the gDNA? I am using phenol/chloroform extractions, then ethanol/sodium acetate precipitation, then ethanol cleanup to extract the DNA.
It's been a long time since I've used phenol/chloroform for this. I now use the Zymo genomic DNA kits, which are very convenient and yield high quality DNA with no dangerous chemistry. They don't advertise the kits for bacterial DNA, but only the lysis step is potentially different.
newtogenetics on Thu Mar 1 23:39:28 2012 said:
I am trying to extract the gDNA from bacteria. If the smaller bands are not plasmids, what else could they be? If they (or the larger bands) are plasmids, at what step would I be losing the gDNA? I am using phenol/chloroform extractions, then ethanol/sodium acetate precipitation, then ethanol cleanup to extract the DNA.
How are you lysing the cells? If you are using an alkaline lysis, then this is where you are losing the gDNA. Note that there are particular pH phenol preparations for each type of DNA and RNA.
I am using proteinase K to lyse the cells. I think the phenol/chloroform I have been using has been equilibriated, and others in the lab have used it for other DNA preps, so I'm not entirely sure it is a problem with the phenol's pH, though maybe if my sample is not at the right pH, then it might not end up in the aqueous layer?
Thanks for all your help!