Ethanol Precipitation

From 2010.igem.org

(Difference between revisions)

Revision as of 20:27, 8 July 2010

Material

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

Procedure

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.
Mix (by inversion or vortex) and freeze overnight in -20° freezer. If you are in a rush you can also put it in the -80 for 30 minutes. 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).
Spin at full speed in a standard microcentrifuge at 4 degrees for 20 - 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.
Wash the DNA with 1 ml 70% Ethanol and spin for 20 min at 4°C. Remove supernatant as before.
Dry the pellet. For this you can air dry (tubes open, ~15 min) or dry in a speedvac. Be careful not to contaminate the DNA by letting anything fall into it. 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. However, overdrying can make DNA hard to re-dissolve. Especially for longer DNA, avoid vacuum drying and airdry only briefly before re-dissolving.
Add your desired quantity of distilled or endonuclease-free water (20 - 50 µl). Vortex and spin down to resuspend.
- NOTE: Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (pH 5 to pH 8.5, for example), 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. 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

Back to Protocol

@@ Line 7: / Line 7: @@
 ==Procedure==
 #Add the following to your sample:
-#*2 volumes of 100% Ethanol
+#*2.5 volumes of 100% Ethanol
 #*1/10 volume of 3M sodium acetate, pH 5.2
-#Mix and freeze overnight in -20° freezer. If you are in a rush you can also put it in the -80 for 3 hours. Dry ice for 10-15 minutes also works.
+#**NOTE: Add sodium acetate first to all your reactions and let incubate for at least one minute. Then add 100% ethanol.
+#Mix (by inversion or vortex) and freeze overnight in -20° freezer. If you are in a rush you can also put it in the -80 for 30 minutes. 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).
-#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.
+#Spin at full speed in a standard microcentrifuge at 4 degrees for 20 - 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.
-#Wash the DNA with 1 ml 70% Ethanol and spin for 15 min at 4°C. Remove supernatant as before.
+#Wash the DNA with 1 ml 70% Ethanol and spin for 20 min at 4°C. Remove supernatant as before.
-#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. However, overdrying can make DNA hard to re-dissolve. Especially for longer DNA, avoid vacuum drying and airdry only briefly before re-dissolving.
+#Dry the pellet. For this you can air dry (tubes open, ~15 min) or dry in a speedvac. Be careful not to contaminate the DNA by letting anything fall into it. 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. However, overdrying can make DNA hard to re-dissolve. Especially for longer DNA, avoid vacuum drying and airdry only briefly before re-dissolving.
-#Add your desired quantity of distilled or endonuclease-free water. Vortex and spin down to resuspend.
+#Add your desired quantity of distilled or endonuclease-free water (20 - 50 µl). Vortex and spin down to resuspend.
 #*NOTE: Beware of using water unless you are sure of what you are getting in to. The "pH" of water can vary widely (pH 5 to pH 8.5, for example), 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. 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.