Ethanol Precipitation

From 2010.igem.org

(Difference between revisions)
(New page: ==Material== * 100% ethanol (for analysis) * sodium acetate * -20°C freezer * Optional: you can use linear polyacrylamide, glycogen or tRNA as precipitation carrier --[[User:Modesto Redre...)
 
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==Material==
==Material==
-
* 100% ethanol (for analysis)
+
* 100% ethanol
-
* sodium acetate
+
* 3M sodium acetate at pH 5.2
-
* -20°C freezer
+
* -20°C or -80°C freezer
-
* Optional: you can use linear polyacrylamide, glycogen or tRNA as precipitation carrier --[[User:Modesto Redrejo Rodriguez|zurdo]] 03:45, 9 October 2009 (EDT)
+
* tabletop centrifuge
* tabletop centrifuge
-
==Procedure==
+
==Procedure: PREFERRED -20°C Protocol==
-
[[Image:Tube placement in centrifuge.jpg|thumb|200px|How to arrange tubes in the centrifuge.]]
+
-
[[Image:DNA EtOH precipitation small pellet.jpg|thumb|200px|Pellets can be very small and hard to see.]]
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#Add the following to your sample:
#Add the following to your sample:
-
#*2-3 volumes of 100% Ethanol
+
#*2.5 volumes of 100% Ethanol
#*1/10 volume of 3M sodium acetate, pH 5.2
#*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 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.--[[User:Etchevers|Heather]]
+
#**NOTE: Add sodium acetate first to all your reactions and let incubate for at least one minute. Then add 100% ethanol.
-
#*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 ˚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).--[[User:Kathmc|Kathleen]]
+
#Mix (by inversion or vortex) and freeze overnight in -20° freezer.
-
#*If you are in a hurry, you can also dip you epi shortly into liquid nitrogen. If you added enough ethanol, the mix won't freeze. Careful with isopropanol - it freezes more quickly. This works well for me and saves me a lengthy incubation in the fridge. --[[User:Jasu|Jasu]]
+
#*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 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.
#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.
+
#Wash the DNA with 1 ml 70% Ethanol and spin for 20 min at 4°C. Remove supernatant as before.
-
#*Overdrying can make DNA hard to re-dissolve. Especially for longer DNA, I avoid vacuum drying and airdry only briefly before re-dissolving. --[[User:Jasu|Jasu]]
+
#Dry the pellet. For this you can air dry (tubes open, ~15 min at 37˚C) or dry in a speedvac for 10 minutes.
-
#Add your desired quantity of water. Vortex and spin down to resuspend.
+
#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.
-
#*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 affect most downstream reactions.--[[User:Kathmc|Kathleen]]
+
#*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==
 +
#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 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).
 +
#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.
 +
#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 at 37˚C) or dry in a speedvac for 10 minutes.
 +
#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==
==Notes==
-
*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. --[[User:JanoschG|Janosch]]
+
Adapted from Ethanol precipitation of nucleic acid protocol on OpenWetWare <br />
 +
Special thanks to OpenWetWare users Heather, Kathleen and Jasu for additional notes
 +
 
 +
Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]]
 +
 
 +
[[Category:Protocol]]

Latest revision as of 19:19, 16 October 2010

Contents

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

Back to Protocol

Retrieved from "http://2010.igem.org/Ethanol_Precipitation"