I want to know the typical conditions for DNA rehydration.
How can I get rid of any remnants of ethanol?
Hi Noha,
Ethanol is volatile, so if you have a pellet it will be white when wet and become transparent when it is dry.
To rehydrate DNA just leave it in buffer TE or water (TE will protect it better because it has EDTA, which will chelate Mg++, which is a cofactor of DNAses, and because it is pH'd at 8.0). If the DNA is fresh, a few minutes is fine. You can improve rehydration by heating at 60-65C for 5 min. If it is extremely dry, leave it with the TE overnight in the fridge.
From the kind of questions you have asked recently you are new to molecular biology. Welcome! I think that it would be great for you to read some basics and learn more, in excellent books and manuals like the 'Molecular Cloning' (see http://www.molecularcloning.com/) or the 'Current Protocols' (http://www.currentprotocols.com/WileyCDA/). These cover basic and very advanced aspects of molecular biology and I am certain you would find them useful in your research.
Cheers,
Ruben
Hi Noha,
Ethanol is volatile, so if you have a pellet it will be white when wet and become transparent when it is dry.
To rehydrate DNA just leave it in buffer TE or water (TE will protect it better because it has EDTA, which will chelate Mg++, which is a cofactor of DNAses, and because it is pH'd at 8.0). If the DNA is fresh, a few minutes is fine. You can improve rehydration by heating at 60-65C for 5 min. If it is extremely dry, leave it with the TE overnight in the fridge.
From the kind of questions you have asked recently you are new to molecular biology. Welcome! I think that it would be great for you to read some basics and learn more, in excellent books and manuals like the 'Molecular Cloning' (see http://www.molecularcloning.com/) or the 'Current Protocols' (http://www.currentprotocols.com/WileyCDA/). These cover basic and very advanced aspects of molecular biology and I am certain you would find them useful in your research.
Cheers,
Ruben
1. To remove the residual of ethanol, you can just air-dry them on the bench (usually I cover them a sheet of aluminum foil to avoid light.). I also place the tubes with DNA pellet inside a tissue-culture hood (we have a hood most of the time is turned on). Since the hood has stronger air flow, the ethanol evaporates faster and the DNA pellet will dry faster. So, I can get my DNA to work sooner.
2. To rehydrate the dried DNA, add autoclaved water or TE (as mentioned by Ruben). Depending on which downstream experiment you want to do later, for Southern analysis, I wouldn't dilute the DNA too much since it needs higher amount of DNA for the assay.
Dear Noha, I agree with previous answers. I should only add an advice: follow the protocols, and write down every detail. If anything goes wrong, you will know what were the conditions (times, temperatures, etc) that you used.
Regarding DNA and ethanol, if you don't let it fully evaporate, your DNA will not be soluble enough, it does not take much time and it gives you reproducible results.
I would do the same as Ruben - let it air dry so the ethanol evaporates and the pellet changes from white to transparent. Then resuspend in a small quantity of pH 8 TE.
I use to rehydrate DNA pellets instead of TE, a solution which consists of 5 mM Tris, pH8 plus 0.1 mM EDTA. The much lower EDTA still protects the DNA but doesn't interfere with any know process such as PCR or ligations. Some ligases are sensitive to EDTA above 0.1 mM.
Try to remove ethanol and maybe change your wash ti isopropanol, it is not as volatile as ethanol is and it quite recommended these days even as a standard step in many kits. If you leave th tube open for a bit (1-2 min) your alcohol is vanishing, your pellet is still there and you should be abe to easily rehydrate your pellet. Follow the kit's instrction for the time of incubation with TE. Having incubated your sample for 0.5 hours instead of 1.5 hours may result in poorer DNA amount in your tube (but that means that you're still going to have some in there-not as much as incubating it for 1.5 hours).
BW
Nikos
We use tris with 0.1 mM EDTA as suggested by Michael David Trus. Then we leave the DNA in the fridge at least overnight to resuspend (may need a little longer)
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