Since our most popular article of all time (“The Basics: How Ethanol Precipitation of DNA and RNA Works”) was published, many of our readers have asked us to further explain the difference between precipitating DNA with ethanol vs. isopropanol and which is the better choice. So today, I’ll meet the challenge and discuss the pros and cons of ethanol vs. isopropanol.
First, let’s review what we know about what is needed for precipitation of DNA or RNA with ethanol:
1. Salt to neutralize the charge on the nucleic acid backbone, causing the DNA to become less hydrophilic and fall out of solution.
2. Ice to chill the sample. Lower temperatures promote the flocculation of the nucleic acids so they form a larger complex that readily pellets under the centrifugal forces of a microcentrifuge.
3. A nucleic acid concentration high enough to force the DNA out of solution (if the conc is not high enough, you can add a carrier nucleic acid or glycogen to enhance the recovery).
4. Centrifugation to pellet the sample
The difference between isopropanol and ethanol is the solubility of DNA in each solvent.
DNA is less soluble in isopropanol so it will fall out of solution faster and at a lower concentration, but the downside is that the salt will too. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at cold temperatures.
DNA falls out of solution in 35% isopropanol and 0.5M salt. Using ethanol, the final concentration needs to be around 75% with 0.5M salt. So for the typical precipitation protocol, isopropanol is added from between 0.7-1 volumes of sample and ethanol is added at 2-2.5 volumes of sample.
The choice of which solvent to use depends largely on the volume of sample you need to precipitate.
If you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice cold ethanol is the preferred choice. You can chill it (some people use liquid nitrogen or -80C to accelerate the precipitation) and precipitate more DNA without the salt contamination that would occur from chilling isopropanol. Afterwards you need to wash the pellet with 70% ethanol to remove salt.
Isopropanol use useful for precipitations where you have a large sample volume (e.g. the eluate you get after using a Qiagen plasmid Maxi Kit) because less solvent is needed, so you can fit the whole lot in the (15ml) tube. But because salts are generally less soluble in isopropanol than in ethanol, they have more of a tendancy to co-precipitate with the DNA. So to lessen the chances of salt precipitation, isopropanol precipitations are carried our at room temperature with minimal incubation times. Once the DNA or RNA pellet is recovered from the isopropanol, you’ll want to wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol with the more volatile ethanol. It is ok to chill the isopropanol precipitated sample, if you are sure that it is not excessively salty.
Because DNA is less soluble in isopropanol, isopropanol allows precipitation of larger species and lower concentrations of nucleic acids than ethanol, especially if you incubate it cold and long. If you do this, just remember to wash the pellet several times in 70% ethanol after pelleting, to reduce the amount of salt you carry over.
So how do you choose when to use isopropanol and when to use ethanol?
Use ethanol if:
- You have room to fit two volumes of ethanol to sample in your tube
- The sample needs to be stored for a long period of time and will be chilled
- You need to precipitate very small pieces of DNA or you have a very low concentration of sample so you want to chill it longer and colder.
Use Isopropanol if:
- You have limited in space in your tube and can fit only 1 volume of sample
- You need large molecular weight species because incubation at room temperature for short periods of time will not be conducive to precipitating small species of nucleic acid
- You are in a hurry and want to accelerate the precipitation of nucleic acids at room temperature
What do I prefer? I use ethanol over isopropanol for most cases, but will use isopropanol if I need to make everything fit in one tube. My preferred protocol is 2 volumes of ethanol and freeze at -20C for at least an hour or overnight for best results. I centrifuge the sample at full speed for 20 minutes to make sure I get everything down. I always wash with 70% ethanol and then centrifuge for 10-15 minutes and keep my eye on the pellet when I decant everything. You need to note or mark the side of the tube where the pellet is expected to be and don’t let it out of your sight when decanting the ethanol!
If I use isopropanol, I avoid cold temperatures because of the excess salt that usually comes down with it. If I want to increase the yields precipitated, I prefer to leave it incubating at room temperature longer vs. chilling the sample. When the DNA is pelleted, the pellet is sometimes more difficult to see compared to the ethanol pellet. It can be clear and glassy. Make sure, again, to note the side of the tube where the pellet should be. Look for it before decanting the isopropanol and 70% ethanol wash. After washing with ethanol, the pellet becomes visible and white. I always make sure it doesn’t slip off the side of the tube wall before decanting the supernatant. Allow the tube to drain upside down for a few minutes and then air dry or speed vac dry (5 minutes is enough) and then resuspend in buffer.
Finally, for dry DNA pellets, heating the sample in buffer at 50-60C will help the DNA dissolve faster and won’t damage the DNA. For RNA, heating can be used too (in water) at temps around 42C. Overdried DNA and RNA will take longer to dissolve so make sure not to speed vac for too long.
So now you know the difference between ethanol and isopropanol and when to use which. If you have any questions, or anything to add, please drop a comment in below.