Team:Northwestern/Protocol/Ethanol Precipitation

From 2010.igem.org

Material

• 100% ethanol
• 3M sodium acetate at pH 5.2
• -20°C or -80°C freezer
• tabletop centrifuge

Procedure: PREFERRED -20°C Protocol

1. Add the following to your sample:
   • 2.5 volumes of 100% Ethanol
   • 1/10 volume of 3M sodium acetate, pH 5.2
     • NOTE: Add sodium acetate first to all your reactions and let incubate for at least one minute. Then add 100% ethanol.
2. Mix (by inversion or vortex) and freeze overnight in -20° freezer.
   • 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. 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).
3. 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.
4. Decant (or carefully pipet off) the supernatant.
5. Wash the DNA with 1 ml 70% Ethanol and spin for 20 min at 4°C. Remove supernatant as before.
6. Dry the pellet. For this you can air dry (tubes open, ~15 min at 37˚C) or dry in a speedvac for 10 minutes.
7. Add your desired quantity of EB (20 - 50 µl); take into account your final desired concentration and the number of reactions you will ultimately want to do. Vortex and spin down to resuspend.
   • NOTE: It has been recommended to perform the 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.

Procedure: QUICKER -80°C Protocol

1. Add the following to your sample:
   • 2.5 volumes of 100% Ethanol
   • 1/10 volume of 3M sodium acetate, pH 5.2
     • NOTE: Add sodium acetate first to all your reactions and let incubate for at least one minute. Then add 100% ethanol.
2. Mix (by inversion or vortex) and put it in the -80°C freezer for one hour. 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. 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).
3. 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.
4. Decant (or carefully pipet off) the supernatant.
5. Wash the DNA with 1 ml 70% Ethanol and spin for 20 min at 4°C. Remove supernatant as before.
6. Dry the pellet. For this you can air dry (tubes open, ~15 min at 37˚C) or dry in a speedvac for 10 minutes.
7. Add your desired quantity of EB (20 - 50 µl); take into account your final desired concentration and the number of reactions you will ultimately want to do. Vortex and spin down to resuspend.
   • NOTE: It has been recommended to perform the 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.

Notes

Adapted from Ethanol precipitation of nucleic acid protocol on OpenWetWare
Special thanks to OpenWetWare users Heather, Kathleen and Jasu for additional notes

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