how to purify plasmid and genomic dna - (Feb/02/2007 )
Really need help here... i want to purify dna plasmid and dna genomic samples, because i wanna use them for real time pcr.
The problem is, i already isolated both genomic and plasmid dna, but afraid that their not 100% pure. I've tried to purify them using phenol:chlroform:isoamylalcohol method, but no traces of pellet can be seen after washing with 100% EtOh and 70% alcohol. Maybe the DNA wasn't there anymore? Noted that I've used the DNA before,and i purified the remaining. is it possible that the DNA has gone? But i thought the DNA has dissolved in TE, should have been left some although i used them right?
If the DNA concentration is too low, Ethanol percipitation will not work. DNA can not be percipitated below a certain concentration (I have not done the experiment to quantify that concentration.) Also it is possible that you may have lost the DNA during the removal of the Ethanol step. Occasionally the DNA pellet may detach from the tube wall, and being so small is accidentally discarded along with the ethanol.
I guess the only thing to do is run a sample of your DNA on a gel and see if it is still there. Be prepared to make a lot more DNA though.
Hmm I thought the % recovery is only like 70-80%.
perneseblue is right.... no point doing ethanol precipitation if too low concentration. Like you said, can't even get enough pellet.
You can precipitate small quantities of nucleic acids. We do viral RNA isolation and we ultracentrifuge our plasma samples, lyse them and add the appropriate amount of isopropanol and also glycogen (this improves precipitation). Even without the glycogen we could precipitate enough RNA to do succesful RT-PCR (on 1/5th of the re-dissolved RNA) starting with 100 copies of RNA, have done so severall times, and the gene we amplified was about 3 kb.