ethanol precipitation-wash with 70% ethanol - (Aug/21/2007 )
Usually, when I wash the DNA precipitate with 70% ethanol, I would add 700ul ethanol first, and 300ul H2O subsequently. It perfoms well when the precipitate is abundant. Although the precipitate would obviously become less after washing with 70% ethanol, I usually owe this to the dissolution of salt. However, when I do the same recently, with a small amount of DNA, I got bad news. Before wash, I can see a weak spot at the bottom of the tube, however it dissappeared after wash. At first, I still thought this may due to the dissolution of salt, which may lead to less amount of precipitation. Unfortunately, I failed in the succedent reaction.
I recalled the detail of my protocol and got puzzled. Is it wrong to add 700ul ethanol first, and 300ul H2O subsequently? I mean, when I add water into ethanol, the water may go down, as the density of water is higher. So, the concentration of ethanol at the bottom, also around the DNA precipitaion, may be much more less than 70% before I mix the liquid. And this may lead to dissolution of DNA into water, and the loss of DNA during ethanol precipitation, since the initial amount of DNA was small??
Sounds reasonable? Can anyone tell me determinately that my protocol (add 700ul ethanol first, and 300ul H2O subsequently) is absolutly wrong? Thank you!
well, I can tell you, that I always have a bottle of 70% EtOH in my -20 freezer for washing DNA pellets - I was told that cold EtOH is better, and I never changed it since it works). I loose only very little during precipitation (a few percent). I have done this with small amounts of DNA also without any problems. Note that the pellets disattaches from the tube quite rapidly, so in order to not loose your DNA, remove the EtOH very quickly (within a couble of minuttes) - but without touching the pellet.
For DNA extraction always keep at -20C: a bottle of absolute alcohol or isopropanol and a bottle of 70% alcohol (both should be use only for DNA and nothing more). For the precipitation add 2-2.5 vol of abs. etoh or isopropanol with the salt (sodium acetate, ammonium acetate, LiCl) and gently mix by invertion (you should see at this point a white precipitate or at least a change in viscosity that's the DNA!!) and leave at -20C overnight of -80C 1h. When centrifuge to get the DNA pellet and washing it with the 70% etoh tap the tube to dislodge the pellet, then centrifuge. Wash at least 2X the pellet. Then dry very well using air dry or a savant (dry with a vacumm). The etoh inhibits many reactions like the pcr.
70% alcohol should be at room temperature to eliminate more salts.
Thanks for your suggestions