Reconstituting plasmid DNA from blotting paper - water vs. TE buffer - (Oct/18/2013 )

Has anyone used water instead of TE buffer to reconstitute plasmid DNA from another lab posted via mail on blotting paper?

What is the downside of using water?

Yes, we had received once from some other lab, and reconstituted in water. Nothing should happen,its DNA.  

water will absorb co2 from the air and acidify. the acidified water will, over a period of time, hydrolyze the dna.

if you will use it immediately then water will be okay. if it will be stored then te (or tris without edta, if necessary) should be used.

mdfenko on Fri Oct 18 12:27:18 2013 said:

water will absorb co2 from the air and acidify. the acidified water will, over a period of time, hydrolyze the dna.

 

if you will use it immediately then water will be okay. if it will be stored then te (or tris without edta, if necessary) should be used.

What is the reason to remove the EDTA from the TE buffer?

Some people think the small amounts of EDTA will affect downstream enzyme activity. This is almost always wrong. The EDTA is present at 1 mM. The fraction of an enzyme reaction composed of the DNA  sample is usually (and desirably) small. So the effective EDTA concentration in most reactions will be 0.1 - 0.3 mM. Enzyme reaction buffers (PCR, restriction enzymes, ligases) typically have 1.5 - 3 mM magnesium. The effect of the small amount of EDTA is insignificant. People who worry about this can use TE with reduced EDTA, 0.5 mM or even 0.1 mM. This is still far better than leaving it out, subjecting your DNA to whatever DNAse contamination is around.

Would you prepare the TE buffer using homemade stocks (Tris and EDTA followed by pH adjustment)? Or is it better to buy a commercially available one? For the peace of mind that the homemade one would not be contaminated with DNAse or enzymes from the autoclave machine or handling.

Either way. 500 mM EDTA is a pain to make, since the EDTA does not go into solution without the addtion of very large amounts of NaOH, and it is easy to overshoot. I'd suggest buying the 500 mM EDTA and making TE. The EDTA makes DNAses unimportant. If you are doing RNA work, then I'd try to buy it, along with most of your reagents.