10 replies to this topic

[AGM1]

Hi,

I have a question about storing DNA long term (>1 year) in water. The DNA I have isolated is bacterial genomic DNA about 40 kb long. A year ago when I ran out the DNA on a gel it was the correct size at 40 kb. The DNA has not been touched since, but was stored in water not buffer. Now when I run the DNA, the situation has changed. There is still the 40 kb band but of less intensity, as well as a large smear from 10 kb to 1 kb.

I have also isolated DNA about 40 kb using a different method 1 year ago, and this DNA has only a smear in the 500 bp-1kb range, and nothing else. This DNA was also stored in water.


Is this smearing and disappearance of bands caused by nuclease degradation or acid hydrolysis due to the water? Or is there some other reason? My thought is that these large pieces of DNA are susceptible to degradation over time due to their size. Any thoughts?

Thanks

[phage434]

There is a reason that DNA needs to be stored in TE.  The Tris controls the pH so that it remains basic, despite CO2 absorbtion from the air.  The EDTA chelates Mg++ ions to disable nucleases which will otherwise chew up your DNA.  Water is not a good storage vehicle for DNA.  In TE, DNA is stable at room temperature for months, although I would not recommend storing it that way.  At -20 or -80, it is stable indefinitely.

[AGM1]

Previoulsy, the DNA was a tight band about 40 kb, now it still has the kb plus the very large smear I mentioned. Do you think degradation of the DNA through either acid hydrolysis or nucleases could have caused the smearing?  Or, would nuclease and acid hydrolysis simply destroy the DNA completely to the point where it could not even be visualized on a gel?

[amitra]

Storage in TE is always the best option. Although from a micro perspective, I have stored genomic DNA in ddH2O for 3 years and never seen any degradation. But I have to say since you still clearly see a band a 40kb, it is still good for any use. Are you diluting your DNA and running it really slow? If you run such a big DNA too fast, you can sometimes get smearing.

[phage434]

I agree that your DNA that still shows a 40Kb band is likely good for whatever you'd like to do with it, though degraded.  FYI it is not really 40 Kb, since gels do not resolve fragments above about 20 Kb (unless they are pulse field gels).  Really long fragments don't move from the wells, so even your original sample was likely partially degraded, to no effect.

[Trof]

I'm bit reluctant to use TE for my DNA since we use it for enzymatic aplications mostly (even though probably the traces of EDTA doesn't have any notable effect) and store my DNA/primers  in 10mM Tris pH8. It's practically a buffered water, so it is "clean" enough and should prevent pH-dependent degradation.
I'm using this since my masters course, when I get mysteriously degraded primers in few months while stored at -20. Never have problem like this since.

[phage434]

This is an unnecessary limitation.  Your enzymatic reactions typically use large amounts of magnesium in their buffers.  If your DNA sample is a small fraction of your total volume, then the impact of EDTA in the buffer is very small, and in my experience negligible.

[AGM1]

I suppose what I was most concerned about was if acid hydrolyis would cause smearing on a gel or if it would just destroy the DNA outright.

[bob1]

It would cause smearing and potentially over a long time cause complete degradation - remember, people have been able to extract DNA from (human) bodies that are several hundred years old - acid hydrolysis is a slow process.

[AGM1]

Perhaps that could explain why my sample still has the intended band at 40 kb, but after a year in water now has some smearing in the 10kb-1kb range.

[Trof]

phage434, on 30 January 2012 - 06:35 AM, said:

This is an unnecessary limitation.  Your enzymatic reactions typically use large amounts of magnesium in their buffers.  If your DNA sample is a small fraction of your total volume, then the impact of EDTA in the buffer is very small, and in my experience negligible.

I never tested the effect of EDTA, but we don't experience any DNA degradation with current storage conditions. In some cases we neeeded to use a large volume of sample, I had a DNA loss in one sample, that resulted in concentration about 2ng/ul, and put a maximum to PCR, but that of course are extremes.