DNA Extraction Problem (No DNA, just a white salt) - (Jun/21/2005 )

I did a DNA extraction today. Up unitl the very end it seemed to be working fine. The Phenol/Chloroform steps all formed a separate layer when introduced and seemed to be taking off the proteins just fine. However, when I added the Ethanol to percipitate out the DNA the whole solution turned cloudy and white. I couldn't find any strings of DNA in the solution, just small white percipitates. I thought at first that maybe the DNA percipitated out with some salt, just there was no DNA visible which has completely confused me. I was wondering if anyone had ever experience this problem and know possibly what went wrong.
Thanks

Just spin down your cloudy solution and you'll get a nice, white pellet which is your DNA.

What organism were you isolating the DNA from? It does sound like the salt is precipitating, you will get DNA as varius said with salts along with it.

Maybe best to resuspend the pellet and then desalt the solution.

Nick

I think maybe there is too much salt in your DNA solution,you can reduce the amount of salt solution during DNA extraction

I guess its protein contamination, you better go for another set of phenol/chloroform extraction and do this step with care so that there won t be any DNA loss and protein contamination, I got such a trouble initially, please update us about your results too after doing the experiment.

cheers
Saint

I´m having the same problem. I work with sponges from museums, and in my DNA extraction appears the same white salt precipitated. I don´t know how this @$#% salt could appear that, but my appraoch in solve this problem is to repeat the 70% etanol washing, centrifuging at 10000 RPM / 10 minutes / 4oC, until the salt (at least almost) disappear.

I´m searching another way to remove that salt, but it´s really salt excess, not another thing, like protein (that ones persist after phenol/chloform step, form cristal arrangement in saturated solution, and are high solubilizable in higher temperatures). Yet, not all specimens present that kind of high salt problem, and this could be an intrinsic variation (or not... how knows).

Any another sugestion will be very helpful. See ya.

My DNA Extraction Protocol is (usually):

*Aprox. 50 ug sample tissue,
*1mL extraction buffer (4M Guanidine hidrochloride, 50mM Tris-HCl pH 8,0, 0,05M EDTA, and 0,5% lauryl sarcosine), bath at 56oC,
*Centrifuge at 3000 RPM for 10 minutes,
*Overnatant with equal volume of Phenol/Chloroform,
*Centrifuge at 3000 RPM for 3 minutes,
*Overnatant with eaquel volume Chloroform,
*Centrifuge at 3000 RPM for 3 minutes,
*Overnatant with equal volume of Isopropanol,
*Centrifuge at 10000 RPM for 10 minutes at 4oC,
*3-4 times (until salt disappear) dry pellet and resuspend with 1 mL 70% Etanol, centrifuging at 10000 RPM for 10 minutes at 4oC,
*Dry pellet and resuspend with 20-60 uL H2O Milli-Q + RNAse (depends on the amount of precipitated)