Can you make nuclease free water or does it need to be bought in? - (Mar/02/2007 )
Can you make nuclease free water – or does it need to be bought in?
....and if so how is it made?
yup you can.
basically one gets standard milli Q water (water that has been desalted by reverse osmosis system. And the most popular of setups come from the MilliQ company, hence the name), add diethylpyrocarbonate (DEPC) to 0.05% final concentration. Shake and incubate overnight at 37 degress. Then autoclave. If you don't like the faint residual smell of ethanol, autoclave the water a second time. And now the water is RNAse free. (protein free)
If you want DNAse free water, autoclaving miliQ water is good enough for that purpose.
For storing DNA fragments, is nuclease-free water better than TE? We have a gel-extraction kit and the last step elutes with nuclease-free water (provided in the kit). But I know that TE is designed to inhibit nucleases (ph8 and the EDTA removes any metal ions), so what's better? Particularly if one considers that after using the DNA fragments for a while it's possible that nucleases might get in anyway.
DNA dissolved in TE is the right way to store it for long periods. Pure water of any kind is a poor choice.
If we store DNA in TE and then use as PCR template, dont you think EDTA will inhibit PCR?
so whats the best choice in this case?
so whats the best choice in this case?
The better choice is to still store your DNA in TE. Accidental nuclease or even plain acidity of water will break down your DNA eventually. So no TE, goodbye DNA and hello reextraction equals much anguish.
The key here is to store your DNA at relatively 'high' concentration in TE. And the fact that PCR is an amazing amplification process.
You don't need much template to get a signal. (hense the reason why cross contamination is a problem to be warry off). Furthermore (at least for pombe genomic DNA), my 'clean DNA' is often contaminated by somekind of residue contamination, I can not remove. If I used my DNA at full concentration, my PCR reactions often do not work. Only if I dilute the DNA at least 1:5 in water, do I get a signal.
So, I keep my DNA stocks in TE. And for PCR, I make 1:5 to 1:25 dilution (in sterile distilled water) of my DNA as working templates.
So I wonder, when I use this kit, should I substitute their nuclease-free water with my TE? I have considered this before.
Yes it is true that TE could inhibit downstream PCR. This is because the EDTA chelates the Mg which the taq needs. But if you do the calculations it is not so bad:
1 mol of EDTA chelates 1 mol of Mg2+
TE is typically 1mM EDTA.
MgCl2 for PCR is usually supplied as 25mM.
This means you would need 25ul of TE to chelate 1ul of MgCl2.
What about using Tris? This will prevent breakdown of DNA by acidity, without having EDTA in there.
For PCR reactions TE is no problem as you only add small amounts of it in your reaction (and indeed, for most pcr reactions you can easily dilute your template in plain water right before the reaction), so the effect would be negligable. But for restrictions or so where you would need more DNA relative to the amount of buffer you add, it might be troublesome to have much EDTA in there.
Using Tris, you should make sure no nuclease contamination happens, but I have never had much trouble.
That's a good point... this may explain some things I have seen in the lab. Although I guess if you have DNA already in TE and you need to digest it you could supplement with more MgCl2. Unless this is bad for enzymes?
I'm not sure which kit you're using - some include an elution buffer which is usually TE. I've used TE to elute my DNA and haven't had any problems.
As mentioned in previous posts, the DNA can be diluted when it's time to perform PCR. Also, I store my PCR primer stocks in TE at a concentration of 100uM and then dilute them before I use them.
As for PCR, most PCR reaction mixes/buffers already contain mag chloride which may negate the TE. As previously mentioned, PCR is a very robust methodolgy. I've done probe - based qPCR with a final concentration of 12 mM and didn't see any deleterious effects.