Which buffers for storing of RNA and DNA? - (Jun/30/2005 )
Which buffer should i use for storing of RNA and DNA?
There are three issues:
* downstream use / convenience
* pH of the buffer
* EDTA / Mg++ concentration
For DNA, I use TE. Usually the DNA concentation is high enough (200 ng/ul) that the dilution of reactions makes the 1mM EDTA inconsequential compared to the Mg++ concentration of the reaction buffer. And the pH is being controlled at a relatively high (8.0 or so) level, where DNA is more stable.
For RNA, the EDTA does no good, since RNAses are active without Mg++. Contol of pH is still important. I use 10 mM Tris.
DNA maxipreps are stored in TE at -20° (aliquot at -80 for long term preservation and future uses) and RNA is stored in RNAse free water (DEPC treated water) at -80°
I don't get this sentence. Should i add EDTA or MG++ or should i add them both?
Would EDTA affect RT-qPCR?
do you mean that i should not add EDTA, but do add Mg++? What is the pH of this buffer?
first question :
EDTA is included in the TE buffer (tris EDTA). what phage 434 means is that for further reactions with these samples, the EDTA included in the TE buffer from this stock solution will not harm your subsequent reaction. For example, if you perform a digestion, it's with Mg++ in the reaction buffer. The EDTA from stock solution is neglectible at this point.
For RNA :
do not add EDTA or Mg++.
EDTA is added in DNA stock solutions to prevent DNase activity. But RNase do not need mg++ to be active. Hence, adding EDTA would not preserve your sample. But in counterpart it can affect other exp you may do with your RNA. I'm not sure, but EDTA in RNA sample would probably affects RT qPCR.
In our lab we just use water for both RNA and DNA without any complications for our downstream applications.
I used Tris-EDTA for my RNA as i used a kit to enrich mRNA and it said to use TE - my RT-PCR failed presumably because the EDTA has chelated the MgCl2 - i use DEPC treated water for my RNA and nuclease free water for my DNA