Tips on ethanol precipitation of nucleic acids and wash - (Mar/13/2005 )

Hi,
another question to this topic:

Does anybody know how much DNA is lost during a "standard" ethanol wash (I use 2 vol 100% EtOH and 1/10 vol sodiumacatate, cool at -20°C for several h, zentrifuge for 20 min at 4°C, remove supernatant, then a second wash with 70% EtOH and same zentrifuge procedure.
Thanks

Peter

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Can I use 100% Ethanol to wash my dna pellets?

Which are the problems if I use 100% Ethanol?

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The only way to know for sure is spec b-4 and after, we never assume 100% and usually expect to get about 90% back, but I suppose you could lose as much as 50%, would depend on the amount of DNA and whether some of the pellet was lost in the proceedure etc...

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Okay first a disclaimer, I am not 100% sure about this...
I think that 100% EtOH will not wash the sample well, you need to exchange the H2O to remove the salt from the sample which is the main reason for the EtOH wash... Also you risk drying out your pellet by removing some of the H2O associated with the DNA and dried pellets are harder to resuspend...

As I say I am not sure of these, hopefully someone else will give their 2 cents...

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hi mario ! late to reply but i hope to clarify some of your doubts !
first think why dna/ Rna precipitates with ethanol? DNA is soluble in a hydrophilic solvent because it interacts with the solvent molecules... non specific and H-bonding. since ethanol has more affinity towards water molecules than DNA/RNA, it breaks the interaction of these two molecules and it itself associates with the water molecule resulting in the precipitation of DNA/RNA (it is equivalent to salting out). capacity to precipitate increases with the " C" no. of the alcohol i.e., methanol< ethanol< propanol. but evaporation decreases with C no. since ethanol can precipitate DNA fairly and evaporates quickly (compared to propanol), it is prefered. some people prefer propanol over ethanol.
also lower the temperatures higher is the precipitation.
1. now coming to your question........ why 70% ethanol? since 70% ethanol contains 30% water, you remove some of the hydrophilic contaminants like salts, soluble proteins, carbohydrate remains,along with water when you precipitate DNA/ RNA.
2. for washing you have to just rinse the pellet with absolute alcohol and pellet down at low speeds. high speeds will compact the pellet and will affect its solubility in the TE solution. also might result in the breakage of the DNA strands.
3. never vortex DNA solutions vigorously, you can just hold the centrifuge tube with DNA and move it in the form of 8but in the horizontal direction.

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Hi everyone,

I'm following a Roche Protocol, the tripure isolation reagent, for DNA extraction from yeast spheroplasts.
After phenol/chloroform extraction, the DNA is first precipited only with EtOH 100%, before an incubation of 5 min. and a 4ºC centrifugation of 2000g 5 min.

1. I'm not quite first if EtOH 100% for 5 min. be enough to precipitate all DNA. Would you reccomend to add NaAC 3M? Besides, it seems to me that the speed of centrifugation is quite slow. Anyway, I've always though..."it is a Roche protocol, it most work well...."

2. This protocol also suggests to wash DNA with three washes of (sodium citrate 0.1M-EtOH 10%)
incubating 30 min with this solution and then pelleting 5 min 2000g at 4º. Does anybody knows how sodium citrate works? Is 10% EtOH and this low centrifugation enough to keep DNA adhered to the tube?

Thanks

Fum

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how much of glycogen is necesary to add in order to precipitate your DNA????, i am having troubles to precipitate viral DNA..........

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hrm...

i always precipitate with 95%+ Isopropyl alcohol, then
wash with 70% EtOH.

and for really low yield preps, i use a product called Pellet Paint. It dyes the pellet pink, so you can track it, and it doesnt interfere with the dna at all.

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Just wanted to add my voice to say that the addition of 1uL glycogen made a huge difference in the recoverability of my plasmid.
I added/mixed just before the addition of 100% EtOH

Happy Holidays
Rob

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Dear All,

I have a question. Our technician maybe left too much of ethanol whlie dissolving the midi prep (probably what stucked on the side of the tube, and later she centrifudeg it, so she got a 2 times more volume than in what she dissolved). Now we want to precipitate the DNA again. We usually use the isopropanol method, but in this case I don't know if ethanol and isopropanol do not disturb each other... so shall we try than the ethanol-sodium acetate portocol? But in this case how can I calculate the needed ethanol volume (for I cannot be sure, how much of ethanol we have now in the solution?)

Cheers

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