Difference between revisions of "Ethanol precipitation of nucleic acids"
|Line 16:||Line 16:|
Revision as of 07:49, 14 September 2006
- To your sample add:
- 3 volumes of 100% Ethanol
- 1/10 volume of 3M sodium acetate, pH 5.2
- Mix and freeze overnight in -20. This step some say is unnecessary but others swear by. If you are in a rush you can also put it in the -80 for a few hours.
- In general, the time you need to incubate in the freezer depends on how much nucleic acid you have and how big it is. My general protocol is to freeze for 20 min to 1 hr at -80 ˚C. This seems to work well for most things, but you may want to freeze longer if you have only a small concentration of nucleic acid or if it is small in size(<15 nucleotides).--Kathleen
- Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes. Make sure to mark the outermost edge of the tube so you can find the pellet easily (or just put the hinge portion of the tube to the outside). It is clear and usually looks like a little smudge on the tube.
- Decant (or carefully pipet off) the supernatant.
- Dry the pellet. For this you can air dry (tubes open, ~15 min) or dry in a speedvac. DNA and RNA (if you don't have RNases in your sample) are typically hearty enough for you to air dry at 37 ˚C, if desired.
- Add your desired quantity of water. Vortex and spin down to resuspend.
- Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (I've seen from pH 5 to pH 8.5), and depurination of DNA at low pH or degradation of RNA at high pH are possibilities. Water also typically contains trace metals, which can accelerate these reactions. I typically recommend resuspension in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). This makes sure your nucleic acid is at a neutral pH and the EDTA will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the EDTA will effect most downstream reactions.--Kathleen
- We tend to wash the DNA with 70% Ethanol after removing the first supernatant (the one containing sodium acetate and 100% Ethanol). Which means add 200-300µl 70% Ethanol to the DNA-pellet. Then spin at full speed for 5mins @ 4°C. Carefully remove supernatant. Proceed with drying. --Janosch