Published: August 12th, 2017 Last Modified: December 6th, 2019
Previously we covered how to reduce cost and waste when it comes to spin kits using silica/chaotropic technologies. However, these types of kits represent the lower end of plasmid purification technologies when it comes to cost and quality of plasmid. Silica based kits, while cheaper, also have a harder time eluting plasmid DNA specifically, and you can often get genomic DNA and RNA contamination, not to mention the possibility of guanadinium salts leftover from sloppy washing.
The next evolution of plasmid kits are based on binding of the DNA to an anion exchange (AE) resin under low salt conditions, washing the resin with a mid salt solution and finally eluting with a high salt solution. These conditions allow separation of plasmid DNA from RNA and other contaminants. After precipitation the quality of DNA is superior to that of silica based kits.
Let’s take a look at the composition of the buffers for two manufacturers of AE kits, Qiagen and Machery Nagel, see how to make them, and talk about packing our own columns!
*** CHECK OUT CHEAT SHEETS FOR EASY REFERENCE ***
While Qiagen and Machery Nagel (MN) both sell AE based kits, there is a slight difference in the composition of the AE resin. Qiagen states that their resin has diethylaminoethanol (DEAE) functional groups, while Machery Nagel uses methylaminoethanol (MAE). Machery Nagel alleges that their technology is optimized towards nucleic acid binding and is therefore…more better? However it’s hard to say without an unbiased side by side comparison.
Both kits work on the same principle of binding/washing/elution with increasing salt concentrations. Take a look at the provided graphs of what molecules drop out at which salt concentrations: Qiagen, Macherey Nagel (pg 11).
So, Qiagen uses NaCl as their salt of choice while MN uses KCl. Surprisingly, Qiagen actually discloses the composition of their AE buffers in their manual, while MN does not, how naughty! However that’s nothing a little patent snooping can’t fix. This patent has the recipes for a kit that is designed to simultaneously remove endotoxins from a plasmid prep, and for whatever reason doesn’t include the recipe for buffer N4-EF, but I think we can figure it out.
As you can see, being built on the same technology the buffers aren’t strikingly different. MAE resin seems to need a lower pH to adequately separate dsDNA from other species, I suppose the curve would look same-same, only different to Qiagen’s.
Since Buffer N4 is not disclosed in the patent, we have to make an educated guess here. I’m willing to bet that since N4 is used as a second wash after N3, it’s purpose is to wash away the detergent from the column. Thus, N4 is N3 without the triton. This buffer would then be equivilent to Qiagen’s QC, which does not contain detergent. For MN’s regular kits you would use N2 to equilibrate, N4 to wash and N5 to elute. FYI, MN calls their regular wash buffer N3 in their non-endotoxin removing kits, but this is likely just N4.
What’s the deal with triton x114 vs x100? Well, seems that x114 has received attention in recent years due to it’s ability to remove endotoxins. MN uses Triton X-114 in their first wash buffer, which preferentially washes away endotoxins while leaving the DNA bound. Qiagen accomplishes the same task in their endotoxin free plasmid kits in a slightly different way. Instead of having the triton in the wash buffer, you add 0.1 volumes of Endotoxin Removal Buffer (750 mM NaCl; 10% Triton X 114; 40 mM MOPS, pH 7.0), ice it for 30 minutes, then load on the column and proceed as usual.
Not so complex or magical when you break them down. Unfortunately, the buffers are only half of what you need if you want to do this type of plasmid purification from scratch. Unlike silica kits, I’ve yet to find a decent supplier of DEAE resin columns by themselves. A dark day will come when I eventually run through my hoard of AE columns. So, looks like we have to start investigating packing our own!
MAE silica resin seems nigh impossible to buy in small quantities, so unless you have some hookups with your HPLC gurus it’s probably not a viable option. DEAE silica resin on the other hand is obtainable. Separation Methods sells 100g of the stuff for ~$500 bucks. Something tells me it’s going to get you far more value than 25 of Qiagen’s maxi columns for $~720.
DEAE sepharose could be a promising alternative as it can be had in smaller quantities. A lovely paper goes into great detail as to how alcohol concentration enhances DNA binding to DEAE resin. Take a look at the buffers the authors use, very similar to the commercial kits.
As a final note it’s worth mentioning that commercial columns can be recycled/regenerated with caution. Older regeneration methods have been proposed, however contamination of the resin with traces of the previously purified plasmid has been reported. The most recent method uses 1M HCl to cleave any DNA stuck to the column. We have used this technique in our lab to regenerate our Qiagen columns 5-10 times with success. This technique would be similarly applicable to DIY columns.
Epoch life sciences sells DEAE midi/maxi columns, can confirm that they work identically (with Qiagen buffers) to Qiagen columns except a third of the price! Sweet! Regeneration with 1M HCl also works. Thank you to the person who recommended it!
PJ’s DEAE Column Regeneration Protocol:
After elution with buffer QF
1. Wash 1X with 1 full column of ddH2O
2. Wash 1X with 1 full column of 1M NaOH
3. Wash 1X with 1 full column of ddH2O
4. Wash 3X with 1M HCl
5. Store in a jar filled with 1M HCl for between an hour and a few months
6. Wash 3X with 1 full column of ddH2O
7. Check that H2O coming out is neutral with pH paper
8. Let air dry, store
9. Equilibriate with buffer QBT, ready for use
There is an alternative to the 1M HCL regeneration method by using 1M Phosphoric acid instead as demonstrated in the following paper. A user reported a degradation in yield of DNA for each regeneration, however, so use with caution: https://www.nature.com/articles/s41598-018-30316-w
Hi,
Your site is very informative. My question is: 1) is there any protocol/method to pack our own deae-silica column? For a plasmid yield of 3-5 mg?
2) is there a way to purify digested fragments from the gel by using the Gel Solubilization Buffer (L3/ QG) and bypassing the column binding step?
3) is there any good method for extracting Viral Plasmids?
I have been stuck here for the past 5-6 months and my work has just slowed down like crazy. I will glad if you can tell me any tips.
Regards
Arjun
Hey buddy,
– This paper is the closest I’ve seen to doing a DIY column, it’s a pretty good read, check it out :https://www.ncbi.nlm.nih.gov/pubmed/12798186
I haven’ tried it as I have old columns I need to use up, but once those run out I’ll give it a go. Another option is to buy a pack of columns from somewhere and regenerate them.
– Are you referring to DEAE columns or silica columns? Generally digested fragments are purified with the silica/guanidine HCL method. What do you mean by bypassing the column binding step? Do you not want the fragment to bind?
– I’ve done a load of work making an infectious DNA clone of a 9.5 kb RNA plant virus, is that the type of viral plasmid you mean? It’s not easy working with large viral plasmids, they can be toxic to E.coli and hard to PCR amplify. Let me know roughly what you’re working with and I’d be happy to help 🙂
My grad students left me with 20 M-N columns and no wash buffer (thanks, kids). I’ll let you (everyone) know how this works.
Thanks for saving my bacon!
Hahaha, those rascals! Probably a case of knocking over the bottle of buffer with an elbow, which I’ve been guilty of myself 🙂 Good luck, let me know how it goes!
FYI, wash buffer for the M-N columns worked like a charm!
Thank you so much for the feedback, I wish you luck in future experiments! 🙂
Great post. I enjoyed reading all of them. Keep the good work.
Just a note: we have been using lab brewed buffer together with Epoch Life Science’s DEAE column ( http://www.epochlifescience.com/Product/SpinColumn/anionexchange.aspx ) for quite a while and it worked really well. Compare to Qiagen’s or MN’s price it’s a bargain. Their silica membrane mini spin column also worked well in our lab with home made buffers ( http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx ).
Never tried to reuse the columns, worrying about contamination. I am aware of publications out there but never able to persuade myself to do so……
Thank you for the kind words, glad you enjoyed the read! Also thank you for the link, I had no idea Epoch sold DEAE columns, that will save us a few bucks for sure!
As for re-using, I know the feeling, just keep the idea in your back pocket in case you ever need it. We regenerate 10+ times with 1M HCl and haven’t noticed issues, however it’s good to be cautious 🙂
I’ve been using Epoch kits for plasmid minipreps and they work very well. I would like to clean and resuse them. Cleaning is easy but do you have recipes for the solutions and a protocol? I assume it would be for the silica minispins. Thanks in advance
Sure, I’ll let you know what works well for me, you’ll need some nice clean water (MilliQ or equivalent) 1M HCl, 1M NaOH and some pH strips.
After you’ve finished eluting your column, run say 30 mL of H2O through the column, follow it up with 1M NaOH, water again then 1M HCl. If you will be re-using them the same day then keep washing with water (3-4X) until the water coming out isn’t acidic (pH strip). If you need to run off somewhere leave the column sitting in a container of 1M HCl, can be stored there for months. When you’re ready to regenerate then wash the columns by putting them in a clean container, filling up with water, decanting the liquid, repeat a few times. Then pass water through the column as before, then equilibrium buffer, and you’re ready to go!
I forgot to add, the 1M NaOH is an extra step I added in, I find that after reusing a DEAE column a few times the flow rate can drop if you don’t clean it out with NaOH, If you don’t filter out all particles after adding solution 3 they can clog the column.
Also once you’ve regenerated your column and run your water through, it’s neutral, etc, you just leave the column to dry somewhere clean and use them whenever.
Can you share the composition of your home brew buffers? We have been using the Epoch kits but would like to switch to using just the columns with homemade buffers.
Thanks!
The Qiagen buffers/protocol works perfectly with the epoch columns 🙂 the recipes are in the back of their manual
Awesome. Thanks!
Hi Jon!
Here is the composition of Qiagen Buffers:
https://openwetware.org/wiki/Qiagen_Buffers
Thanks! Great link.
For preparation of the Endotoxin Removal Buffer (QIAgen formulation), what is the most appropriate grade of MOPS to use? Cheers.
I would feel like anything above 98%, 99% purity will do just fine for the vast majority of applications. Consider that you’re washing away recently deceased ecoli through your column, I don’t think 99.5% vs 99% or 98% will do too much. Find a reasonable grade that you’re comfortable feeding into your downstream application and is affordable, and don’t be afraid to shop around, sigma isn’t the be all end all.
Thank you for all your kind advice. I have problem with finding MN’s patent about MAE resin. Can you send it to me in email ？My email is [email protected]. Thank you very much.
Email sent!
But yeah, sorry but I haven’t found any MN patent explicitly using/synthesizing MAE resin, in the following patent https://patents.google.com/patent/US6428703 they reveal a few buffer recipes and the fact they use nucleobond columns, but that’s it 🙁
They also mention that they use MAE resin on their website and manuals, but that’s it. If I ever find a synthesis for MAE resin I’ll add it here.
https://www.nature.com/articles/s41598-018-30316-w
I just encountered this since I have overspent on my funding (setting up a new lab is costly here due to exchange rates and stuff…) and I need regeneration of whatever I am using. This paper (Sci Reports from China) postulates that 1M phosphoric acid works better than 1M HCl, also with a shorter procedure (20 mins tops) for mini columns.
I will be trying this on the MN NucleoPure Xtra’s that I have (I usually save them just in case). I’ll keep you posted 🙂
I’ll give phosphoric acid a go, so far I’ve been using 1M HCl with very good success! Columns can be regenerated at least ten times, in our experience. The catch is that you need to make sure that your lysate is free from debris of the solution 1-2-3 lysis, the particles can clog the resin and ruin your yield.
I use two layers of fisherbrand filter paper and a funnel to clear the lysate, vs the expensive Qiagen filter columns.
Phosphoric Acid works wonders, is much faster to do and essentially cleans both silica gel and AE columns. I have used one silica gel column (MN NucleoSpin) the 10th time with no reduction in performance and no cross-contamination. Only will use fresh columns for stuff to be transfected or transducted from now on.
Thanks for the advice, I’ll update the post with the new information!
Hi Alex,
I had some MN midi columns that I have regenerated via the H3PO4 procedure and I have used the buffers that you have provided here (waited months for X114…) and obtained excellent results with regenerated MN columns. One thing though, in the patent the pH of the last buffer (elution, N5) was given as 7.0 – just to give you a heads-up about that.
Take care,
ML
Glad it worked for you! As always, thank you for your input and correction, I’ll fix it right up.
Hi there
a couple of month a go I’m pretty sure I found a protocol that integrated separating endotoxin during plasmid purification on silica based spin columns using triton x-114 ! I cant find it any more, do you guys have a protocol for this?
Also do you guys have an idea what is the difference between the endotoxin removal buffer on the midi plus and giga prep kits for qiagene. Is the components in the ETR buffer different from the table you have put here?
cheers
I just fell curious that mini spin columns are very cheap in China market.
We can buy 0.1$/ one spin columns. No one reuse it here.
And you can make it for youself. Whatman GF/B glass fiber membrane,
Punch it to make circle. Buy empty plastic tube and tool for punch circle from China.
and Whatman GF/B glass fiber membrane, you can make it
and you fell trouble, Just buy spin columns directly from China. 15$/100 Preps for RNA， DNA， plasmid like epoch. I know lots of spin columns sold to USA by OEM. if you have interest.
I am not sure I can shareinfo here？
Yeah, you can share suppliers if you like! I’ve had good experiences with suppliers from China, it’s good to get some experience negotiating with the manufacturers
Enzymax and Epoch likely get them in bulk straight from China anyway, they’re just a closer reseller for us. I don’t think most scientists know they can go straight to the source for some of their reagents.
I’ve been doing the 1M HCl regeneration of my columns for years. I generate a lot of acid waste. Do you know if the 1M HCl can be used multiple times? Not knowing for sure I only use it once.
Also, for clearing the lysate after solutions 1+2+3, I put filter floss (Carolina Biological Supply) in a 60cc plastic syringe until it is about 1/3-1/2 full. I pour the lysate into the syringe and let it sit ~5minutes then put in the plunger and force the aqueous solution out. I treat the syringes with HCl also. After many uses, the plungers resist insertion so I discard them and get new ones. The tip of the syringe must be plugged while the lysate sits. For this I am fortunate to have many caps for QIAfilter cartridges from Qiagen HiSpeed Kits.
Yep, if the claim of the HCl cleaving DNA down to nucleotides is true, then at the worst you’ll have to wash away some nucleotides before you pass the equilibriation buffer through. I have my columns sit in a tub with 1M HCl until I feel like regenerating a bunch, so they’re sharing the 1M HCl. I usually make a bit of fresh stuff when regenerating, but that can go back in your tub.
Thanks for the tip about the DIY filter, that’s very helpful! I’ve been using two thin filter papers and some miracloth…the Qiagen lysate clearing filters are hilariously expensive.
Hi, I like the M-N midi kit because of the filter paperstep at the beginning makes it faster than qiagen kits. Do you know what that filter is made off of? Is therea too for a DIY equivalent system?
Great site, Thank you for the pieces of advice
It’s funny, I’ve actually been playing around with exactly this…paying 10-20$ for an expensive syringe filter doesn’t sit well with me.
Basically the material I’ve been looking at is filter felt, you can get it in pore sizes as small as 0.5 micron. It’s used alot in the aquarium industry.
The tricky bit is cutting the filter in a shape that fits the syringe and doesn’t let anything past it, you need a few layers. I would probably follow it up with a cheapy 0.2 micron syringe filter, total cost probably less than 2 bucks including the syringe? Needs some work, cutting polyester felt isn’t as easy as you’d think!
I have seen a suggestion using coffee filters but have not yet tried it.
https://bitesizebio.com/8688/how-to-to-speed-up-commercial-midi-and-maxi-plasmid-preps/
Yep, this is my current go-to method for maxi’s. One layer of mira cloth on top of two coffee filters. The mira cloth gets the big chunks and drains fast, the coffee filters get the small stuff. I set up a funnel inside a 125mL flask, pour in the goop, lift up the miracloth and all the fine stuff drains into the coffee filters. Not as fast as the expensive syringe filters but cheap cheap cheap!
Just coffee filter also works fine, the only extra thing is to pass 5-6 mL’s of the equilibration buffer after passing the lysed bacteria through the filter instead of squeezing it so that the excess DNA drips away. I have used the same culture (divided into two parts) with both fresh MN midi columns (with the fancy filter) and regenerated MN columns with coffee filter and I have observed no discernible loss between two setups. Saved a lot of money by doing this 🙂
Hi, I have a bunch of older Qiagen Tip 20’s. They were intended for plasmid purification but are they the same as Qiagen’s Tip 20/G sold for genomic DNA preps? IOW, are they both the same DEAE resin-silica resin? I need to do genomic preps and will just use the older Tip 20’s if there’s no difference. Thx from a fellow jockey : )
Thanks for the post! Can I use Qiagen buffer for M-N column to purify EndoFree plasmid?
Thanks for the informative post!
I’m currently using the MN nucleobond xtra midi kits, which are fairly economical (£5 ea). The endotoxin free ones are more than twice the price however. I was wondering if I can make my own endo wash buffer?
The recipes above are a great start, as I already have triton x-114 from manual endotoxin removal. However, looking at the protocol there is also a FIL wash buffer? Is there some triton in the equilibration buffer of the standard kit anyway, to reduce endotoxin?
nucleobond standard kit: https://www.takarabio.com/documents/User%20Manual/NucleoBond%20Xtra%20Plasmid%20DNA%20Purification%20User%20Manual%20%28PT4011/NucleoBond%20Xtra%20Plasmid%20DNA%20Purification%20User%20Manual%20%28PT4011-1%29_Rev_12.pdf
nucleobond endo free kit: https://www.mn-net.com/media/pdf/18/eb/8f/Instruction-NucleoBond-Xtra-EF.pdf
Hmm, so I think the purpose of the FIL buffer is to wash the remainder of your lysate from their filter syringe, and likely to wash the resin a bit as well. The MSDS does not mention any alcohol present, and it does not say to add ethanol/isopropanol to the FIL wash buffer. My guess would be that it is enough KCl to allow binding to the column (900mM) and enough Tris for buffering to a low pH (100mm pH 6.3). So basically equilibration buffer without the ethanol or Triton.
Hello PJ,
I enjoyed learning the inner workings of the various DNA prep kits. Similar to one of the earlier corresponders, I would like to know the composition of the BB buffer and ETR rinse solution in the Qiagen Midi “Plasmid Plus” kits. Also, do you know the column material in those kits? One of their patents is very vague, but I suspect that it is a porous silica bead, as the capacity is much higher (at least 10 X) than that of the glass fiber filter pads. Finally, Zymo makes a similar midi kit, but the endotoxin removal is a second column, as opposed to a Triton X-114 rinse. However, in my experience, you loose a lot of DNA going over that second column. Do you know what the two Zymo columns are made of?
Thanks!
Hello,
I’m currently trying to prepare Endotoxin Removal Buffer using Triton X-114.
I first dissolve NaCl (750 mM) and MOPS (40 mM) in 80 mL ddH2O , adjust pH to 7.05 by NaOH, and then add 10 mL Triton X-114. But after adding Triton X-114, the solution becomes cloudly and seems that something is not able to properly dissolve.
May I ask what’s the solvent used to prepare the Endotoxin Removal Buffer?
Should I add isopropanol to 15%?
Thanks!
Hello mentors
I am a beginner of the DIY Maxi kit. My name is Tomo.
I was using the FavorPrep™ Plasmid DNA Extraction Maxi Kit.
http://www.favorgen.com/port_h1.php?type_1=h_v_search
The yield of DNA was quite good. However, the cost is the problem. After the elusion, plasmid will be processed to the precipitation. It looks anion exchange column.
Then, as the next step, I purchased Enzymax Maxi spin column.
https://www.enzymax.net/columns/maxi_column_DNA_RNA.htm
The procedure for the extraction we exactly followed the protocol form Enzymax. We used 5X PE for wash.
https://www.enzymax.net/columns/maxi%20column%20DNA%20prep.pdf
Enzymax looks very thin matrix compared with favorgen. The problem is the final elution step. Even if we drained 2ml of elution buffer, only 1ml can be recovered. The yield of the plasmid is around half of favorgen.
My question is,
1) The principle of plasmid extraction of favorgen and Enzymax is different? Why Enzymax column bed is so thin?
2) Is there nay alternative way to improve the plasmid yield of Enzymax?
3) I checked the price of Epoch. Around $10 is a little bit expensive since the currency exchange ratio becomes much worse in recent day. Thank you for your help.
Dear Mentors
I would like to buy the Epoch 2040-050 Anion Exchange Midi & Maxi Columns from directly manufacture. Epoch is a just seller of the manufacture probably in China. Do you have any information about manufacture?
Hello, I have been searching for the best recipe for plasmid extraction using silica columns for several months. I have tried many protocols and made different types of solutions (along with the mentioned items on this site), but unfortunately, the final concentration was not sufficient (about 40-50 μg/mL). I would be grateful if you could help me make suitable solutions to achieve a higher concentration of plasmids and optimize the process.