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DIY Phase Separating Gel: Clean and Cheap!

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Phenol/chloroform extractions are a common lab technique to remove proteins from aqueous solutions containing DNA and RNA.  They can be tedious and a bit time consuming if you are working with a lot of samples.  And accidental carryover of phenol/chloroform can inhibit downstream applications.

Over time, I thought I had achieved some level of competency in removing most of the aqueous layer without getting any phenol or protein gunk in my sample.  But there is no harm in trying to perfect your technique, is there?

When I read Jode’s article on phenol/chloroform extraction, the section on Phase Lock gel piqued my interest as it was something I had not encountered before. Essentially it is a gel that is denser then water, but less dense than buffer saturated phenol/chloroform. When centrifuged with the latter two reagents, it migrates between the phases, “locking” the organic phase at the bottom. The aqueous phase is then easily removed.

This sounded pretty great, but shelling out close to $200 for 200 samples (that’s a dollar a sample!) worth of gel seemed a little steep, so I began looking for alternatives. Turns out, other Bitesize Bio readers were thinking along the same lines. Doug commented that Sigma used to hold the patent, under the name “Phase divider gel”. Roberto found a very informative paper in which researchers found that using vacuum grease in phenol cleanups produced a nice interphase without degrading RNA.

I am an adventurous sort of guy and we just happened to have several jars of Beckman vacuum grease lying around the lab. After searching through some MSDSs, I managed to glean that the vacuum grease is composed of polydimethylsiloxane (dimethicone) and silicone dioxide. I also managed to find a scribbled on MSDS for Phase Lock gel that described it as a “Proprietary Mixture” of CAS#: 63148-62-9 and 112945-52-5. Not surprisingly this also works out to be a mixture of polydimethylsiloxane and silicone dioxide (I got you now, Phase lock gel!).

Armed with that knowledge I decided to give it a try! I scooped some vacuum grease into a syringe, autoclaved it and squirted some of the gel (approximately 50-100 µl) into an eppendorf tube and spun for 5 minutes at max speed to pellet the grease.  I then added my RNA-containing solution, phenol/chloroform/isoamyl alcohol and spun the mixture for 5-10 minutes at 16,000xg.

It worked excellently – the phases were well separated after centrifugation and there was little degradation of the RNA (as compared to a sample cleaned up without grease). I began using it routinely and quickly ran out…but I wanted MORE!

Next, I tried to mix up my own gel. I bought some polydimethylsiloxane (DM 350) from a cosmetic supply company and silicone dioxide from a spice store and I set to work.  At first I tried  85% DM350 and 15% silicone dioxide, roughly the same proportions cited in the MSDS of different types of vacuum grease.  Although it worked decently well (the gel migrated into the middle as expected) a bit of the liquid DM350 seemed to float on top of the gel and granules of silicone dioxide migrated down into the organic phase. I believe the trouble lies in obtaining polydimethylsiloxane with a high enough viscosity and silicone dioxide with a fine enough mesh size to keep the layer intact. I kept experimenting with different types of polydimethylsiloxane but they all failed.

At that point I broke down and bought a big ole’ tube of Dow Corning High Vacuum Grease off eBay. It’s the same stuff cited in the paper posted by Roberto. 150 grams cost me $30 shipped. I figure the entire tube should be good for 1000-1500 extractions in 1.5 mL tubes (assuming I use about 100 mg (50-100 µL) of grease per tube.   That’s only pennies per sample!

The Dow grease worked as well as the Beckman grease and has the added benefit of coming in a tube that can be easily squirted into a syringe.

So learn from my adventures, save yourself some money, buy a big tube of vacuum grease and enjoy the nice phase separations!

13 Comments

  1. Alec on August 9, 2016 at 9:17 am

    Hi! Great post. Do you know what the density of the gel is? Strangely, my aqueous phase always gets trapped in between two layers of gel. The lowest layer is organic, then gel, then aqueous, then gel, and nothing on top. The DNA extraction buffer I’m using has a pretty high concentration of salts which sets the density to around 1.29 g/cm^3. Do you think this would cause the solution to migrate in between the gel?

  2. Jayson on July 26, 2016 at 11:51 pm

    How much of the Dow Corning High Vacuum Grease would you use for a 50ml-tube format?

    Thanks

    Jayson

    • Cyril on June 1, 2017 at 3:49 pm

      Hi Jayson,
      Did you find the rigth volume of Dow Corning High Vacuum Grease for a 50ml-tube format ?
      Cheers,
      Cyril.

  3. Alex on March 17, 2016 at 7:55 pm

    I bought some corning vacuum grease and tried this method myself. It works great for the classic phenol-chloroform-isoamyl alcohol extraction, however when you try to use the same grease with TRIZOL reagent, the grease settles on top of the aqueous layer. I think this may be due to the large amount of guanidinium salt in the TRIZOL reagent. This salt makes adding salt to the subsequent isopropanol precipitation reaction unnecessary, however in this case it causes the aqueous layer to be slightly denser than the vacuum grease.

    From my experience with phase-lock gels, they usually come in “light” and “heavy” versions. The “heavy” version is what is recommended for TRIZOL extractions. I assume “heavy” is code for “denser than water with salt in it”. It’d be interesting to try different brands of vacuum grease to see if any of them would qualify as “heavy”, and thus work well with TRIZOL.

    Thanks for this post. Recently all phase-lock gels were discontinued, so I was looking for an alternative. You came to my rescue!

    Best,
    -Alex

    • Romain on March 24, 2016 at 10:32 am

      Hi,
      I’m interesting by the answer too.
      Is someone test a formulation to mimic the heavy lock gel?
      Thanks!

    • sumira on May 12, 2016 at 8:51 pm

      Alex, did you ever discover what to use in liu of phase lock gel heavy? I am in the same boat, trying to isolate both RNA and DNA from trizol. Thanks in advance for any advice!

  4. Fabio on February 18, 2016 at 5:50 pm

    Hey Alexander,
    thank you very much for your tip! Just tried the Dow Corning High Vacuum Grease and it works perfectly.
    Cheers,
    Fabio

  5. Basak on February 10, 2016 at 2:18 pm

    Hello!

    I tried this for DNA extraction during the chloroform extraction phase, but with all the dissolved stuff in the water phase, the gel was actually less dense and it stayed on the top. I could still get the water phase out with a syringe, but just wanted to say that it is worthwhile making sure the the density of your water phase will be right, otherwise the gel doesn’t to to the interphase.

  6. Ana Patricia Ramos on April 30, 2015 at 11:06 am

    This is great ! Phase lock gels are crazily expensive, so I will give this a try 😉
    Thanks for sharing

  7. JacoboReyes Velasco on October 11, 2014 at 8:49 pm

    Hello Alexander,

    Thank you very much for this article. I am working with some very old tissue samples with low DNA yields and increasing the amount of DNA I can get is extremely important. Quick question, do any type of syringe works for autoclaving? or did you use a special syringe? also, for how long and at what temperature did you autoclave it?

    Thanks!

    Jacobo

    • Alexander Klenov on April 8, 2015 at 8:37 pm

      Hey Jacobo,

      Any type of syringe will work as long as it withstands autoclaving. I autoclaved the syringes at 121C for 30 minutes.

      Cheers,
      Alex

      • Magnus on April 26, 2016 at 6:46 am

        Hi, What is the purpose of autoclaving the gel? Does it impact the physical properties of the gel or is it simply to sterilize it?

        Thanks
        Magnus

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