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Recycle Those DNA Extraction Columns

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You know those ridiculously priced and throw-away DNA mini, midi and maxi-prep columns? Well the good news is that you can actually re-use them if you are reasonably careful at regenerating them, with this simple and cheap method described in detail by Nagadenahalli B. Siddappa in Biotechniques in 2007.

Apparently these columns can be reused up to 20 times… perhaps more a guesstimate than a real number, but hey, who’s complaining?

Essentially the columns are treated with 1M HCl overnight, rinsed extensively with dH2O and then re-equilibrated with buffer from the kit you use (e.g. Qiagen QBT). If you are worried about finishing the buffers in the kits, look in the back of the manual, there are often simple recipes about how to remake them.

The authors allay concerns about plasmid carryover contamination by assaying for plasmid using RT-PCR and transfection assays which all showed zero carryover between recycled uses of the columns. In fact they showed that the plasmid DNA had been chemically sheared into low molecular weight fragments and could not be used as a template.

They also stated that prolonged exposure to 1M HCl (1 month) did nothing to the binding capacity of the columns, so even if you forget about it, there is no need to stress.

Our lab has modified the protocol to include passing warm water through the treated columns when regenerating to ensure that all DNA and any other contaminants are removed. Also pre-warming the elution buffer to 42oC or thereabouts seems to increase the elution of any plasmid DNA bound to the column, although what extra compounds it releases is unknown but probably negligible.

It may be our imagination, but we have often gotten higher yields of plasmid DNA from a treated reused column, than a new one.

Go ahead, stretch those budgets further.

27 Comments

  1. Albert on November 8, 2017 at 3:35 pm

    Hi,
    I would thank if somebody can give me a recipe of an spin column activation solution for DNA purification.
    Thanks !

  2. Krzysztof Treder on January 18, 2017 at 12:22 pm

    Jode,
    Maybe for regenerated Quiagen silica columns, buffers described by Boom et al. (1990) can be used instead of proprietary N3, PB and QG? They were designed to purify DNA/RNA using silica particles or diatom earth, so should work the same on silica in the columns. Just guessing.
    Cool protocol anyway.

  3. Kanomi Sasaki-Capela on July 14, 2016 at 12:11 am

    Hello Everyone,

    The Qiagen manual states that the max yield for a midi prep is 100ug. I just performed a midi and ended up with 183ug of DNA. Normally a high yield is a good thing, but 183ug is unusually high. Has anyone yielded more than the described 100ug for a plasmid midi prep? Or is it possible some kind of contamination gave me a higher O.D. than the actual amount?

    Thanks,
    Kanomi

    • deemah dabbagh on September 28, 2016 at 9:49 pm

      Hi,

      I have never used the midi kit, but I have used qiagen’s mini and maxi kits and have ended up with conentrations as high as 300 ug/ml with the mini and 500 ug/ml with the maxi both with good quality DNA.

      Best,

      Deemah

      • deemah dabbagh on September 28, 2016 at 9:50 pm

        And the OD was in the correct range both of the times.

    • Ruchita Selot on December 8, 2016 at 1:28 pm

      Hi Kanomi,
      I have used the midi kit and gotten plasmid yield of 220ug from one column. However, it gave poor transfection result so obviously the forced elution is messing up with the DNA! Have to figure out another way of getting the high concentration of DNA without compromising on the quality…
      Regards
      Ruchita, Bangalore

  4. Desanka Maksimovic on November 12, 2010 at 2:13 pm

    Hello everyone!

    I’m using Sigma’s Spectrum Total RNA Kit. For the binding columns, I have found the protocol for recycling, but I’m wondering what to do with the filtration columns. I have an impression that they can’t be reused. Can I do the RNA instraction without them?

    Thanks

  5. Christ Fotiadis on October 8, 2010 at 6:31 am

    Hello to all of you

    I want to ask a question about the re-used columns. Did anybody tried to digest the plasmid from that columns? I tried a lot of times to digest it, but it was unable to digest. When i use a fresh new column with the same plasmid and digest it all goes well. I use the re-used columns for pcr without any problem. Do you know something about it? Thank You and sorry about my english.

    • Prashant on March 19, 2019 at 7:53 pm

      Could you elaborate “unable to digest”?

  6. Liam on April 15, 2010 at 3:34 pm

    Hi Bill

    This depends on your column. I have only used the Qiagen columns, although the Macherey-Nagel ones are probably the same. These are gravity based, but I don’t see why you couldn’t use vacuum filtration (I use vacuum filtration to clean them). I think you would just need to test it on equal volumes of the same prep to see if it is suitable.

    Good luck

  7. Bill on April 15, 2010 at 3:02 pm

    Can I use vacuum manifold instead of gravity flow during extraction?

  8. Steve on November 18, 2009 at 3:51 pm

    Thanks for the detailed reply. I am trying out the silica method on some qiagen miniprep spin columns and will see how it goes. we also have peqlab miniprep tubes which I will look at. I will post my results…

  9. Jode on November 17, 2009 at 5:22 am

    I’ve been using this method for a while, and I’ve tried it on both the gravity-based DEAE columns (like the Maxi Prep) and the silica membrane based spin columns from Qiagen. There are Mini-Prep scale columns for both, and I have the feeling from some of the posts that they are being confused.

    The DEAE based columns (the DNA elutes in high salt and must be precipitated afterwards) work great with this system. Like Liam, I’ve found that the capacity of the columns actually increases after regeneration, and I’ve been re-using the same columns for well over 20 times with no detectable degradation of the columns.

    Here is the protocol that I worked out by sampling the flow-through of the column washes onto litmus paper. I would encourage anyone who is doing this to test the protocol with litmus paper for yourself the first time you try it. When I refer to column volume here, I don’t mean the bed volume of the resin, but the actual maximum volume that the plastic column will hold (ex. 30 ml for a Q500 (Maxi), 2.75 ml for a Q20 (Mini)).
    – Drain the 1M HCl stored in the column
    – Wash with 1 column volume of Sterile, Nano-Pure (18M?) Water (NPW)
    – Wash again with 1 column volume of NPW
    – Wash with 1 column volume of DNA Elution Buffer (Buffer QF)
    – Wash with ½ column volume of NPW
    – Equilibrate with 1 column volume of Equilibration Buffer (Buffer QBT)
    Perform the DNA prep according to protocol.
    – After eluting the DNA, wash the column with an additional ½ column volume of Elution Buffer
    – Wash the column twice with 1 column volume of NPW
    – Wash the column with ½ column volume of 1M HCl
    – Cap the column with a luer cap, add ½ column volume 1M HCl, and parafilm the top.

    Occasionally, when I pull a column out of storage I find that there are gas bubbles in the resin. When I see this, I wash the column once with NPW to get rid of the majority of the acid, then cap the bottom securely and fill the column halfway full with NPW, then insert a rubber stopper with a single hole in the top of the column and apply a vacuum with my aspirator. I rap the column gently against the edge of the counter until air stops bubbling out of the resin, then I release the vacuum, un-stopper the top, uncap the bottom, and allow the column to drain. I then continue the protocol outlined above with the second column volume of NPW. I’ve found I can do this with Q100 on up, but when I tried it with a Q20 it just sucked air through the capped bottom. (I’m currently experimenting with ethanol washes to eliminate the gas bubbles instead of vacuum treatment.)

    I found that I can regenerate the silica based spin columns as well, but they aren’t as robust to repeated uses. I stored the columns with 500ul of 1M HCl in them, with the top capped with a 1.5ml Eppendorf Tube cap. This wasn’t perfect, and sometimes the columns dried out, but this didn’t seem to negatively affect the column. To use the columns I would do the following:
    – Spin the columns at max g for 30 seconds to drain the acid
    – Wash with 800ul of 100mM Tris (pH 8.0) twice
    Perform the miniprep according to protocol, and elute the DNA
    – Return the column to the 2ml wash collection tube and wash with 800ul TE
    – Wash the column with 800ul of NPW
    – Wash the column with 200ul 1M HCl
    – Add 500ul to the column and cap the top, store in the 2ml collection tube

    I found that the columns could be re-used several times without any loss of DNA binding capacity, but eventually the membrane in the bottom of the spin column would start to detach from the sides and pieces of it would come off during the wash steps. Since this made me nervous, and I didn’t want to try to re-invent the Qiagen buffers N3, PB and QG (all of which are proprietary), I decided to discontinue regenerating the spin columns.

    • Alex on August 8, 2016 at 11:37 am

      JODE,

      Thanks for the clarification. I have some questions related with the silica-based spin columns. You mention a step: „Return the column to the 2ml wash collection tube and wash with 800ul TE“. What does TE mean? After how many uses did the membrane start to come off from the spin column?

      Also, since , I have a low copy plasmid to be isolated, I need lot of material. For this purpose I would like to use the same column twice for the same bacterial suspension, since I think it is a better idea to use the column twice than to pellet lets say 10 ml bacterial suspension in one round. An I have only a miniprep. Do I need to wash the column in between? If yes, can I use the same washing step as for the repeated use of the columns described by you? Since I do not really want to spend the buffers from the kit for this purpose.

      Thanks a lot,

      Alex

  10. Steve on November 16, 2009 at 1:19 pm

    did anybody come up with a definitive protocol for this, including the re-equilibriating in buffer? Is washing 5 times in dH2O sufficient to reactivate the martix or does it require washing in buffer (e.g buffer p1 or elution buffer from qiagen kit for example)?

    Thanks for any assistance.

  11. Liam on February 20, 2009 at 7:09 am

    I usually was them 3 or 4 times in distilled water to help get rid of salts, then pull through half a column of HCl (1M) and place the column in a 5L beaker, so it is submersed. They soak overnight, then get 5 washes the next day, allowed to dry, and reused.

    Most miniprep kits do give good enough DNA for sequencing, however I have used the recycled columns, and good old 1 2 3 and have had no problems. It is mostly determined by the hands of the user.

  12. Jerry on August 28, 2008 at 9:52 pm

    Thanks for the tip, I was using it for a while, but I am a little confused here. The article on BioTechnique was not clear to me, could you please make an order what have you done to regenerate those columns. (e.g 1-wash dH2O 5times, 2-Wash with washing buffer 2 times etc…)

    And how about Invitrogen columns, has anybody tried them?

    Thanks in advance!

    -J

  13. Nick on April 16, 2008 at 11:14 am

    Hi Liam

    LIC is certainly reliable enough for generating constructs for protein expression.

    It’s a bit different from TA cloning – the principle is explained in this article: https://bitesizebio.com/2008/01/08/get-your-clone-90-of-the-time-with-ligation-independent-cloning/

  14. bala on April 16, 2008 at 9:48 am

    Nice tip,I’ve tried this with atleast mini-prep columns – and the columns get damaged! probably because they’re cheap ones! a few of my friends say that this method works great with Maxi kit columns, would try that out and post the outcome.

  15. Liam on April 16, 2008 at 9:18 am

    For the cleaning of the columns, I recommend a vacuum manifold, e.g. like the one from Promega, it makes the cleaning of these columns very quick. We convinced them to give us one in exchange for using their maxiprep kits, which we now recycle.

  16. Liam on April 16, 2008 at 7:16 am

    Nick

    I was wondering, not having read the ligation independent cloning technique in detail (it is in effect TA cloning of sorts is it not ?), as to whether it can be used, or whether it is reliable enough to generate DNA constructs for protein expression, where everything needs to be in frame ?

    Liam

  17. Nick on April 15, 2008 at 9:17 pm

    Hi Patrick. Drop me an email using the contact form so we can talk about that.

  18. Patrick on April 15, 2008 at 7:03 pm

    Cool, I was about to drop you an e-mail and suggest this as a futur article.

    Btw, I’m still up for the ligation indepedent cloning vector project

  19. Liam on April 15, 2008 at 3:10 pm

    With the miniprep columns, I would be very careful putting them in any stirring solution, the matrix is not the same as in the bigger columns, perhaps just a good soak would be sufficient, then again, perhaps good old soln 1 2 and 3 is sufficient for screening.

  20. Chad on April 15, 2008 at 3:00 pm

    Well, looks like I know what I’m going to be doing with my down time this week.

  21. Kyle on February 27, 2009 at 12:42 am

    I definitely want to start doing this in my lab, and just have a couple of questions so I do this right, both pertaining to the final steps. I haven’t worked with HCl much (besides in my stomach), but I know it’s not something you want to play with and obviously needs to be gone from the preps. My first question is since the entire prep soaks in HCl overnight, what’s the best way to clean it off, inside and out (squirt it with water, dip it in water, etc)? Secondly, I don’t use a Qiagen kit with QBT buffer, I use the pcr and spin minipreps, and am currently in the process in changing from Qiagen to Zymo. I’ve found the recipe for the re-equilibrating QBT, but I don’t think a component of my kits resembles that recipe. So if it matters, should I use a solutions from my kits (if so, which one) or make me some QBT and use it? Sorry for the density, and thanks in advance.

    • Stefy on November 1, 2016 at 9:58 pm

      Hi,
      How did it go with switching to the Zymo columns? I had but it did not go that well for me. I’m wondering if someone else had the same experience or if it was that I’m just a newb

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