Simple ethanol precipitation was a disaster and have no idea why! - (Sep/19/2009 )

I would be very grateful if anyone has any ideas that could help me understand what happened to some of my DNA samples, as this was the last step of a long process with some important samples and I'm quite gutted it went wrong (I guess most people know that feeling!).

The samples I precipitated were 6kb PCR products which had been gel extracted using the stratagene gel extraction kit (Gelred used instead of ETBr). There was 20ug (pooled from several pcr reactions) of each sample in 300 ul of elution buffer. Samples had been quantified using a picogreen assay and a small amount run on an agarose gel, which agreed with the pico green and showed nice clear 6kb bands and no degradation following the extraction. So everything appeared fine prior the the ethanol precipitation.

So I added 1/10 vol of 3M sodium acetate pH5.2 (from sigma DNase free) and 2.5 time new total volume ethanol. Mixed by inverting 20 time and left in the -20 for 30 min. Spun for 20 min at max 14000xg. Could see pellet in all the 12 tubes. Carefully poured away supernatant (but kept it separately for each sample and have not thrown it away). Washed with 0.5ml of 75% ethanol (retained that too). Took great care not to disturb pellets and am confident I didn't lose them, as could see them clearly. Allowed them to dry for 5 -10 min, so not over dryed. Added appropriated amount of water to make them 120ng/ul, based on the earlier quantification, using the pico green assay. Heated then to 65C for ten min to dissolve and mixed by flicking the tubes, then on ice.
Then re-assayed them by pico green and now 4 of the 12 samples have only about 4ug of DNA and not the 20ug i expected and the others have between 11 and 15 ug. Ran a small amount on a gel the 4 samples with low DNA yield form two faint bands one 6kb and the other approx 3kb, and a little tiny bit of smear (the other are a single band of the correct 6kb size).

Then checked the concentration of the samples on a nano drop and they all had similar concentrations of about 150ng/ul according to the nanodrop. The 4 samples have 260/280 of 1.55 and the other sample are a bit higher 1.6-1.8.

I can not figure out what happened to 15ug of DNA lost from each of these samples, in one simple ethanol precipitation???
Have tried
1. Re spinning the ethanol supernatants for 2 hours after storing them overnight in the freezer, no pellet. May need more salt if the dna is there?
2. Thought maybe the samples had become single stranded, as then the may not have shown up well with intercalating dyes such as gelred or pico green, but would still by measured on the nanodrop. So with one of the poor samples I heated it to 95 and then cooled it slowing (0.2C per second), hoping it would reanneal, if these was the case. Ran a small bit on a gel and now the two faint bands have gone and there is nothing!
3 Only other ideas are that some sort of chemical, maybe from the gel column, is contaminating the samples and then when I heated the to 65 it destroyed the dna? Or may intercalating dyes can no longer intercalate, because the dna damaged? Not sure why the other samples seem ok.

I think I'm going to have repeat the experiment, but would really like to understand what went wrong.
Thanks in advance for any help.

I vote for the water you have dissolved the pellet in. I wouldn't use water, I'd use TE. Water of low quality can contain lots of things, such as heavy metal ions. At 65C, those things can trash DNA pretty effectively (let alone at 95, as you have seen). You should not need to heat the DNA to dissolve it, especially if it has not dried. Water can also have low pH, which will cause problems in dissolving DNA. The EDTA helps in chelating heavy metal ions.

I would also make sure that all of the DNA was precipitated from the ethanol solution. Add another 20% ethanol to your supernatents, chill for a while, and then spin down. The ethanol at 2.5x may not be quite enough. Also, make sure the wash in 70% ethanol is really 70% ethanol, not a little less, which can happen if some evaporates.

You might consider pellet paint next time you have valuable samples you are precipitating, since it makes the pellet visible and unmistakable.

If I remember right this picogreen measurement is quite temperature dependent, have you measured your samples at an appropriate temperature, i.e. 20°C? Otherwise there might me a great error in the measurements.
I'd add some glycogen to the precipitation, that helps the DNA to precipitate...
And with TE and avoiding 65°C during dissolving again I agree. I use always low TE (10,0.1) and let the DNA dissolve at room temperature.

phage434 on Sep 19 2009, 08:51 PM said:

Thanks for the comments it is good to know others think along the same lines as me. The water was nuclease free water from sigma and I used a new aliquot. I have frequently heated DNA to 65 without problems (and they have been to much higher temperatures many times during the original PCR). Also prior to loading them on the gel I carried out ethanol precipitation on these same samples, to concentrate them and they were fine, the only difference I can think of is that there would have been more salts and other stuff present from the PCR reaction, previously.
Ethanol was 75% and made up freshly and I also tried adding a bit more ethanol too, prior to spinning it again.

Didn't want to add anything else to the samples, as they are to be sent away for deep sequencing and also 20ug of DNA should be easily visible and that was the case. Although one thing I noticed was a slight hint of orange and am wondering if some of the gel red came through. Thanks again for your idea and this at least helps me realise there is no obvious way of savaging them. I have a suspicion that the gel extraction columns may have become compromise and leak something into the sample,when eluted. The gel fragments were quite bit and so were dissolve in a large quantity of gel extraction buffer and so it took several application to get it all on the column.

What I could try is taking a small amount of my sample and mixing it with some other DNA, heating it and seeing if the other DNA is destroyed. This would at least tell me if there was something damaging in my solution. But then if it was been degraded surely i'd get a smear and not two weak bands.

hobglobin on Sep 19 2009, 09:08 PM said:

Thanks for the reply. I think the pico green was ok, as standard curve was fine and it was at room temperature, about 20. Also pico green result correlated with the intensity of the bands on the gel. Although neither of these matched the nanodrop readings which suggested all the sample contained similar amounts and lots of DNA, but uv based measurements do tend to overestimate, as any contaminant that contributes to the 260 will make it appear more concentrated than it really is. And the poor sample had the worst ratios.
I will use TE in future, as I agree it is probably safer, but as mentioned above these same samples went through ethanol precipitation by the same method and were fine.

As we say in our lab, it's called research and not just search!

keliang8 on Sep 21 2009, 05:21 AM said:

Hi and thanks, I realise that is often the case, but the stratagene kit seem to return a good yield of 70% when I previously tested it (gel quantification). Besides, I don't believe it was loss during the gel extration as I checked the samples by pico green and agarose gel and they were there and appeared fine, after the gel extraction, prior to the final precipitation.

So, it is still a big mystery

The other thing you have added to the sample is your 3 M NaOAc. I could imagine it has become contaminated with nucleases or heavy ions. If you continue to be interested in this problem, I would suggest trying to precipitate a known amount of some known-good DNA with the same NaOAc and water, followed by washing with the same ethanol (which could also be the source of the problem). I still think it is heavy metals in your water/NaOAc/ethanol. There is a reason for the EDTA in TE.

phage434 on Sep 21 2009, 01:05 PM said:

That would seem likely but the other 8 samples proscessed at the same time with the same solutions were ok, so surely these would be destroyed unless it was a borderline thing. And if it was nucleases or heavy metals then surely there would be a smear. Will use TE in future, but suspect something was damaged on those particular columns which for some reason didn't damage the dna until I ethanol precipitated them and heated them.
Thanks again for your thoughts, especially as I'm keen for this not to happen again.