TITLE: Ethanol precipitation of RNA from small or large volumes
AUTHORS: Stephen Floor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to precipitate RNA with ethanol and resuspend it! Two separate protocols - one for small and one for large volumes. The procedure for large volume precipitation is similar, but begins with an initial precipitation and transfers to a small tube for the final steps to avoid loss in the large tube. Strongly recommended to move to a smaller tube.  </div><div class = "text-block"><span>Recommended reading: Walker & Lorsch </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">RNA purification--precipitation methods</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Precipitating RNA (< 500 ul sample volume)]
Add 1 ul glycoblue per 250 ul sample volume
volume here is the volume after adding NaOAc, not the original volume.
?. [Precipitating RNA (< 500 ul sample volume)]
Vortex briefly
?. [Precipitating RNA (< 500 ul sample volume)]
Add 2 volumes 100% ethanol
volume here is the volume after NaOAc, not the original volume. For example, add 1 ml ethanol to 500 ul NaOAc solution.
?. [Precipitating RNA (< 500 ul sample volume)]
Vortex briefly
?. [Precipitating RNA (< 500 ul sample volume)]
Incubate at -80 degrees for at least 1 hour or in a dry ice ethanol bath for 30 minutes.
[PAUSE] RNA is more stable for long periods when stored in ethanol at low temperature than as a pellet. Shorter incubation times are probably ok - glycoblue protocol recommends 30 minutes at -20 C.
?. [Precipitating RNA (< 500 ul sample volume)]
Pellet RNA at  16k g for 25 minutes at 4 °C
Tip: orient the tubes so the hinge is pointed outwards and the pellet will be easier to identify along the hinge-side of the tube.
?. [Precipitating RNA (< 500 ul sample volume)]
Aspirate supernatant carefully without dislodging pellet, first using a P-1000 for most volume and then a P-20 for the end. Using a vacuum aspirator is not recommended as it is easy to aspirate the pellet.
?. [Precipitating RNA (< 500 ul sample volume)]
Gently add 500 ul of 70% ethanol at -20 °C
the warmer the 70% ethanol is, the more RNA will re-solubilize. Take the 70% out from the -20 right before adding it and then return it to the -20.
?. [Precipitating RNA (< 500 ul sample volume)]
Pellet RNA at  16k g for 5 minutes at 4 °C
?. [Precipitating RNA (< 500 ul sample volume)]
Aspirate supernatant carefully without dislodging pellet, first using a P-200 for most volume and then a P-20 for the end.
?. [Precipitating RNA (< 500 ul sample volume)]
Quick spin to bring down remaining ethanol
?. [Precipitating RNA (< 500 ul sample volume)]
Remove as much ethanol as possible without aspirating pellet using a P-20
?. [Precipitating RNA (< 500 ul sample volume)]
Dry for 5 minutes at RT and not longer - fully dried pellets are harder to resuspend
?. [Precipitating RNA (< 500 ul sample volume)]
Add the desired volume of water or buffer
?. [Precipitating RNA (< 500 ul sample volume)]
Scrape the tube along a tube rack to dislodge pellet
?. [Precipitating RNA (< 500 ul sample volume)]
Quick spin
?. [Precipitating RNA (< 500 ul sample volume)]
Measure concentration using nanodrop or qubit
?. [Precipitation of RNA (large sample volume)]
Add NaOAc to sample to a final concentration of 0.3M
This can be accomplished by diluting the sample by at least five volumes with 0.3M NaOAc or adding 3M NaOAc to a final concentration of 0.3M.
?. [Precipitation of RNA (large sample volume)]
Add 1 ul glycoblue per 250 ul sample volume (after adding NaOAc)
?. [Precipitation of RNA (large sample volume)]
Vortex briefly
?. [Precipitation of RNA (large sample volume)]
Add 2 volumes 100% ethanol (1 ml 100% ethanol to 500 ul NaOAc-diluted sample)
?. [Precipitation of RNA (large sample volume)]
Vortex briefly
?. [Precipitation of RNA (large sample volume)]
Incubate at -80 degrees 1 hour to overnight.
[PAUSE] RNA is more stable for long periods when stored in ethanol at low temperature than as a pellet.
?. [Pelleting and resuspension of RNA]
Centrifuge in a conical tube at > ~5000g, for 25 minutes
?. [Pelleting and resuspension of RNA]
Aspirate supernatant carefully with a serological pipet or vacuum aspirator
don't worry about getting all the ethanol off - trying to get all the ethanol off will likely remove some pellet as this is a loose pellet
?. [Pelleting and resuspension of RNA]
Add 750 ul of 100% ethanol at -20 °C
?. [Pelleting and resuspension of RNA]
Resuspend chunks of pellet in the 100% ethanol by pipetting up and down
?. [Pelleting and resuspension of RNA]
Transfer ethanol and chunks to a microfuge tube
?. [Pelleting and resuspension of RNA]
Spin for 5 minutes at 16k g at 4 °C
?. [Pelleting and resuspension of RNA]
to wash and resuspend pellet
?. [Precipitating RNA (< 500 ul sample volume)]
Add NaOAc pH 5.2 to sample to a final concentration of 0.3M
This can be accomplished by diluting the sample by at least five volumes with 0.3M NaOAc or adding 3M NaOAc to a final concentration of 0.3M.[CRITICAL] Do not exceed 500 ul NaOAc-diluted volume per 1.5 ml tube. For large volumes, see alternate protocol below.
?. [Precipitating RNA (< 500 ul sample volume)]
Record the sample volume. If unknown, you can estimate it using the side of the tube or meausure it by pipetting it.