Dear Mark

I am not quite sure whether this is really all salt what you see but I suspect that you transfer some proteins from the interphase. Perhaps you should repeat the PC step once more. Sometimes PC dissolve material out of the plastic tubes and then it´s looking milky, too.

In normal cases the pellet should be a more or less transparent substance at the bottom, like you describe it in the step without salt. Alternatively you can use one volume isopropanol instead of 2 vol ethanol for the precipitation. We usually saw threads of DNA after precipitation when harvesting the DNA of a 60mm dish, which could be fished with a peg or a pipette tip. After isopropanol precipitation the washing is done in 70% ethanol.

Actually I would recommend a switch to purification columns, phenol is very aggressive and in Germany the disposal of such special waste is extreme expensive. In the meantime I avoid it if possible.

Wash your pellet with 70% ethanol. DNA is not soluble at this condition but the salts washed out. After adding 70% ethanol, gently break the pellet to re-suspend it. Then spin it hard to re-pellet the DNA. This works pretty good.

Dr. Addlagatta,

Thank you for the kind reply. I am sure to include a 70% ethanol wash during my precipitation protocol, but I still am left with this very salty pellet. Another odd thing is that when I try to re-precipitate in a lower volume, I am able to get the white cloudy solution to clear by heating to 65 degrees for 10 minutes... however, when I check the concentration it has not increased. It seems to be stuck at a maximum of 300ng/ul

Dear Mark

Without salt You will not be able to precipitate nucleic acid, however, You can try another salts. Ammonium Acetate, for instance. I can not promise that it will help, but You can try to prepare the saturated solution, cool it down several hours under -20C, and add 1/10 volume of not-frozen upper part. Also cool down EtOH before adding. After precipitation wash the pellet with 70% EtOH twice.

By the way, what 260/280 ratio did You get?

Regrading re-precipitation in the smaller volume, please note that You are loosing some of nucleic acid after each re-precipitation.

Hope it will help. If not, I would try another method of extraction.

Dear Mark

I am not quite sure whether this is really all salt what you see but I suspect that you transfer some proteins from the interphase. Perhaps you should repeat the PC step once more. Sometimes PC dissolve material out of the plastic tubes and then it´s looking milky, too.

In normal cases the pellet should be a more or less transparent substance at the bottom, like you describe it in the step without salt. Alternatively you can use one volume isopropanol instead of 2 vol ethanol for the precipitation. We usually saw threads of DNA after precipitation when harvesting the DNA of a 60mm dish, which could be fished with a peg or a pipette tip. After isopropanol precipitation the washing is done in 70% ethanol.

Actually I would recommend a switch to purification columns, phenol is very aggressive and in Germany the disposal of such special waste is extreme expensive. In the meantime I avoid it if possible.

I agree with Michel Marcus, even after washing with 70% alcohol still ppt exist, mostly likely it's protein . So carefully give PC treatment to remove proteins.

To remove protein, add 1 volume 5M ammonium acetate without ethanol and spin 10 minutes at 15K x g. Abs 260/280 should be 1.8 @ pH 8.

If ur 260/280 values look good, then 70% ethanol wash should solve the problem

Thank you everyone for the very helpful advice. I will take aliquots of my samples to try each suggestions until I figure out what the problem is. My adviser looked at it and said it appears to be salt or detergent made it through the phenol:chloroform extraction and has formed a DNA-salt-detergent complex of some sort. We're not sure the best way to get rid of this (or if it is even possible)

I used a protocol from the Molecular Cloning handbook which stated to use 0.5M EDTA with 100ug/ml proteinase K and 0.5% Sarcosyl for digestion of cellular proteins. I think the EDTA and Sarcosyl have made it through the ethanol precipitation as she advised.

Once I try everyones suggestions I will report back with the results. Thank you again!!

I was able to try every suggestion listed here (except for purification column because I dont think we have one) but the contaminated DNA extractions remain as such. I tried additionally a cleaning with sodium perchlorate which helped slightly but not enough to salvage the DNA. It appears to be catastrophic detergent / EDTA contamination.. I tried a different protocol using much less EDTA (5mM instead of 500mM) and the results were much better - shimmery strands of DNA were present, my final concentration was much higher (500-3000ng/ul) and the 260/230 ratios were above 2.00

To summarize, I need to make sure the protocol makes sense before using it even if it was published!

Thank you to all of the scientists above that offered suggestive help.

What you saw was EDTA. The original protocol used dialysis to removed 0.5M EDTA.

Sorry I didn't notice the EDTA concentration. BTW, there are many misprints in methods sections. Good Luck!

Precipitate it without the NaOAc. Try lowering ith concentration from 3M to 1M. May be it should help.

Use 7.5-10M ammonium acetate (add 1/2 volume) instead of NaOAc. Both ammonia and acetic acid will evaporate in speed vac.

I made the same observation: After precipitation, sodium acetate form a white cloudy pellet on top of the DNA. I agree with Jai:Precipitation without sodium acetate works better. If the amount of DNA you try to pellet is limited, add glycogen before you add ethanol.

Hopefully the OP has found his answer by now. I was having the same issue (low 260/230 ratio) when precipitating genomic DNA with isopropanol and NaOAc. I solved it by replacing NaOAc with NaCl to a final concentration of .3 M. Increasing the concentration to .5 M negatively affected the ratio. I also tried the precipitation with 100% Ethanol and NaCl and the results were bad as well.