Tips for sequencing miniprep DNA - (Oct/28/2006 )
I have a few questions about some minipreps that I'd like to sequence.
I followed the old-fashioned alkaline lysis procedure from Maniatis, and RNased the final product. The plasmid is about 3.7 kilobases (pGEM T-easy plus insert).
1. The absorbance at 230 - 260 - 280 indicate that I have very little protein (260/280 = 2) and that I have a lot of DNA (about 50 micrograms DNA from my 3 ml culture.) Is that a normal yield? It seems very high to me. One of my preps gave me several hundred micrograms of DNA from a 3 ml culture. !! This can't be right, can it? I don't see any RNA on a gel, so I don't think that RNA is responsible for the high reading. ?
I looked at the DNA on a gel, and it looks generally good and robust and cuts easily.
2. I resuspended the DNA in TE. Should I worry that the EDTA might inhibit the sequencing reaction? It will be at a final concentration of about 0.1 mM.
3. Phage recommended 300 ng of DNA for a sequencing reaction. Since we're just starting to sequence in the lab, using an automated sequencer, I am tempted to try a curve with one sample, to see what sort of range of miniprep volume/DNA concentration we can use and still get good sequence. I am considering 100ng plasmid DNA, 300 ng, and 1 microgram. This would correspond to about 2, 6, and 10 microliters of the miniprep per sequencing reaction. Does this seem reasonable?
thanks for any advice you may be able to offer!
50 micrograms DNA from a 3 ml culture seemed quite high to me. normally, one can yield up to 30micrograms from a miniprep. 260/280 ratio tells you how clean is your DNA. and it is considered ok when the ratio is in between 1.65 and 2 as far as i remember. 1.85 is the best ratio i think.
wow, very impressive. Really getting alot of DNA from your miniprep.
Try to use TAE buffer with reduced EDTA if you got. But I think EDTA is only affecting the PCR reaction. Perhaps not for sequencing.
I believe it is unlikely that you truly have 30 ug of DNA from a 3 ml culture. I would recommend testing that by running a gel -- and not believe a spectrophotometer reading. You may have a lot of DNA, but it may be genomic contamination rather than plasmid DNA. Or, it could be phenol contamination.
EDTA at high levels will inhibit sequencing reactions, by chelating the Mg++ required. But this is rarely a problem at the concentrations of DNA normally encountered, where a 300 ng/ul sample is diluted 1:12 in a sequencing reaction into water. Since the Mg++ is normally at 2-5 mM in a PCR or sequencing reaction, the 0.1 mM (final) EDTA will have little effect on the reaction.
Thank you one and all!