Material
- 100% ethanol (for analysis)
- 70% ethanol (stored @ 4°C)
- 3M sodium acetate pH 5.2 (store @ 4°C)
- 5M ammonium acetate
- -20°C freezer
- Optional: you can use linear polyacrylamide, glycogen, or tRNA as a precipitation carrier
- tabletop centrifuge
Procedure
Eppendorf Protocol
How to arrange tubes in the centrifuge.
Pellets can be very small and hard to see.
- Add the following to your sample in the order they appear:
- 1/10 volume of 3M sodium acetate, pH 5.2, or 1/2 volume of 5M ammonium acetate
- 2-3 volumes of 100% Ethanol
- Mix and freeze overnight at -20. This step some say is unnecessary but others swear by it. If you are in a rush you can also put it in the -80 for ten minutes to a few hours. Dry ice for 10-15 minutes also works.
- In general, the time you need to incubate in the freezer depends on how much nucleic acid you have, how big it is, and the volume it is in. My general protocol is to freeze for 20 min to 1 hr at -80 ËšC. 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).–
- If you are in a hurry, you can also dip your epi shortly into liquid nitrogen. If you add enough ethanol, the mix won’t freeze. Be careful with isopropanol – it freezes more quickly. This works well for me and saves me a lengthy incubation in the fridge.
- 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 speed vac. DNA and RNA (if you don’t have RNases in your sample) are typically hearty enough for you to air dry at 37 ËšC if desired.
- Overdrying can make DNA hard to re-dissolve. Especially for longer DNA, I avoid vacuum drying and air dry only briefly before re-dissolving.
- 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 into. 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 affect most downstream reactions.
96 Well Plate Protocol
- Add to each 10 µl product:
- 1.9 µl of Na acetate 3M
- 60 µl of 85% ethanol
- Mix thoroughly (vortex ???) and keep at -20°C for 30 min
- centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)
- remove supernatant (invert tube on trash once)
- add 150 µl of 70% ethanol and mix
- centrifuge for 15 min at 4000 rpm and 4°C
- remove all supernatant (invert tube on trash)
- invert the tube on paper tissue
- centrifuge for 2 min at 500 rpm
- take out the tube and let it dry in the fume hood at room temperature for 10-15 min
- put 20 µl of formamide dye using a multi pipette (nasty chemical to manipulate in fume hood)
- vortex thoroughly/spin/vortex/spin
- transfer the 20 µl (multi pipette) to a sequencing plate
- put septum on top, press, tap once (do NOT mix)
- keep on ice in the aluminum rack
- Heat for 3 min at 95°C (SWATI/95-CST) to keep in single-stranded form)
- keep on ice in the aluminum rack
Notes
- We tend to wash the DNA with 70% Ethanol after removing the first supernatant (the one containing sodium acetate and 100% Ethanol). This means adding 200-300µl 70% Ethanol to the DNA pellet. Then spin at full speed for 5mins @ 4°C. Carefully remove supernatant. Proceed with drying.