The storage buffer of the RiboLock RNase reagent contains 50% (v/v) glycerol that prevents the overdrying of RNA. My question is what if we would use ethanol along with glycerol for DNA drying too.
Тhank you.
I'm not quite sure what you are asking? Are you currently having problems with overdrying your DNA pellet?
Samantha Jeschonek: sometimes. I just wondered, what if we would be using mixed ethanol and glycerol to decrease the risk of overdrying DNA. In order to no worry about this problem.
I was wondering about the possibility of storage of DNA in glycerol and googled.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020885/
Roeder et al. 2010 compared in this study the storage of DNA in
- TrisHCl buffer,
- TrisHCl buffer + 50% glycerol,
- distilled water + 50% glycerol
100 days storage, at temperatures:
- 4 degrees,
- 0 degrees, and
- -20 degrees celsius.
Their result:
The least changes occured when DNA was frozen at
- -20 degrees celsius in
- distilled water + 50% glycerol!
(1xPCR buffer - Tris HCl did not enhance the storage).
They cite
Schaudien, Baumgärtner, and Herden 2009
whoconcluded in their study that glycerol prevented ice crystal formation.
So glycerol might be used to preserve DNA.
However, in our human genetics institute, we stored DNA at 4 degrees in TrisHCl (genomic DNA, so higher concentration several hundred nanogram/microliter or around 1 microgram/microliter - diluted DNA didn't last as long ... ) which is a standard.
What kind of overdrying do you mean? After extraction step in 70% Ethanol? Or just storage?
We prevented overdrying by wrapping tightly the closed eppendorf tube with parafilm for long term storage.
Like it is shown in figure 1-2 D and E http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch01s02.html
(but with a closed 1.5 ml Eppendorf tube). This always helped to seal the closed Eppendorf cups - enough to prevent any kind of drying.
Overdrying after DNA extraction, I prevented by acceleration of the drying procedure in the tube, by putting the DNA pellet in the Eppendorf tube with a last drop of ethanol into a heating block at around 50 degrees celsius (or less). Removing of most of the Ethanol using a 100 microliter and a 20 microliter pipette accelerated the drying much more. And then it is a matter of just few minutes. And I was standing these minutes next to the block and eye-checked again and again until the pellet is not too dry but also not wet. Before dissolving the DNA pellet in TrisHCl or distilled water.
Thank you, Gwang-Jin Kim!
We store DNA in 1xTE buffer in -20*C.
>What kind of overdrying do you mean? After extraction step in 70% Ethanol? Or just storage?
I will try to be more precise: as you know, after extraction step in 70% Ethanol we need to dry our DNA, but sometimes our DNA becomes overdried and insoluble. So, my question is what if we mix ethanol and glycerol to avoid overdrying of our DNA after "70% Ethanol extraction" step. I think if we mix glycerol and ethanol, our DNA will not be overdried because its will be with glycerol. Thus, you no need to stand and scrupulously eye-check again and again until the pellet will be ready.
In this way, it is interesting that we may use some mix glycerol-based solutions to store DNA. I think that this information is very useful. But I'm not a chemist and I don't know what may occur during mixing ethanol, glycerol, and DNA. Don't we get some DNA adductors or whether inhibits glycerol PCR?
Not sure in as much glycerol inhibits PCR.
It seems that in small amounts, it is not inhibiting.
Just found http://www.bio.net/mm/methods/1996-February/039703.html googling...
I guess, the higher the concentration of your DNA during storage, the more stable is the DNA.
And if you take out some little amount of it for PCR, then the glycerol concentration won't be that high in your final PCR reagent. So a high concentration of the DNA would be desirable for your matter.
But I think the overdrying you can really avoid by wrapping your closed tube using parafilm. Or using special tubes for long term DNA storage such like
https://www.fishersci.com/shop/products/thermo-scientific-nalgene-general-long-term-storage-cryogenic-tubes-nalgene-system-100-cryogenic-tube-sterile-1-0ml-ext-thread-conical-bottom/033377x
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