Tuesday, January 13, 2009

Cheap Minipreps Now!!!!

Here in the Bay we purify a heck of a lot of plasmid DNA using over-priced commercial kits. Mainly because there's a fully stocked Qiagen biobar down the hall and this set-up appeals to our excessive laziness. But sometimes even laziness can have its limits. Maybe the Ottawa transit strikes and blizzards are getting to me. Whatever the reason, today I was particularly struck by the ridiculousness of shelling out hundreds of dollars of grant money for a week's supply of aqueous solutions and mass-produced plastic tubes, packaged in totally unnecessary cardboard and throwaway Nalgene bottles. Surely a Ph.D. biochemist can come up with something more efficient than this?

The simple two-step procedure used by Qiagen and most other commercial plasmid prep kits is no secret. First comes Birnboim's alkaline lysis - a series of three buffers that lyse the bacteria and then neutralize the lysis reaction. Second, plasmid DNA is purified from the lysate by passing it over a silica column in the presence of high salt. Making the buffers is trivial (see below). Plus, we probably have a lifetime's supply of unused buffers from previous Qiagen kits lying around the lab. So we're paying for the little plastic columns. About $1.60 CAD a pop. Making the columns is probably not much fun, and alternative materials don't seem to be as efficient. But in theory there's no reason the columns can't be re-used.

The idea of regenerating commercial miniprep columns first came to my attention a while back. This paper/ad described a two-buffer product called MaxxBond that allowed columns to be regenerated and re-used up to ten times. I liked the idea of recycling columns, but not the idea of relying on yet another over-priced, proprietary/patented technology of an unknown nature. I was particularly concerned about the possibility of unknowingly cross-contaminating my plasmid preps, which would be a total nightmare. It's impossible to assess the risk of this happening without knowing how the columns were being regenerated.

After a little discussion in the Bay today, a quick surf on the Google produced a better alternative now floating around the interweb. The Indian research group of Ranga describe a method that involves simply storing the columns in hydrochloric acid and rinsing them with water when you're ready to use them again. They claim commercial columns can be re-used 10-20 times in this way. What I find appealing about this method is that the HCl acid works by physically destroying residual nucleic acid from your last prep, so it would seem pretty much impossible to cross-contaminate the next one. And a 10-fold increase in column productivity sounds pretty darn nice, and drops that price from $1.60 to 16 cents per prep. I think I might actually try it.

The protocol is available here, and more info can be found in a paper here.



12 comments:

rob said...

Nice job on following up blabbing in the bay.
The Bayblab saves you money!

Anonymous said...

The Indian HCl method works well, but their claim that you can indefinitely store the columns in acid is false.

More than a week in 0.1 N HCl will start to soften the silica matrix. The top surface becomes warped and in some instances the packing will fall through the bottom of the support with sufficient vacuum pressure or centrifugation.

It's better to wash your used column with water, spin it dry, and keep it in a cold dry place until it's ready for a 12-hour treatment with 1 N HCl.

Kamel said...

Dude,
Nice post! The proprietary-ness and super disposability of those kits is annoying. But you don't need to use the columns at all. We always used the Birnboim method, followed by phenol-chloroform extraction/precipitation. Not a Qiagen kit in sight (unless we're talking maxiprep).

Now we need to find a way to save on those expensive precast acrylamide gels... (yeah, yeah, pour your own)

Anonymous Coward said...

yeah I tried re-using an agarose gel once just for fun. It doesn't work. Even if you re-dip into EtBr solution. I'm not sure why, but the DNA doesn't run properly...

Bayman said...

@anon,

Thanks for the tip. I wondered whether the HCl would eventually melt the plastic. I'll go with overnight or an hour instead of long-term in the acid.

Bayman said...

I also wonder there's an acid-proof way to mark how many times each tube has been re-used.

Anonymous said...

H&E pen can survive etoh and formalin, and I doubt graphite will be erased by hcl

Anonymous said...

Even regular sharpie won't come off in the acid (to my frustration, since all the columns eventually become covered in multiple people's writing). I bet VWR marker should work fine too.

Alejandro Montenegro-Montero said...

On a related matter....How much are we supposed to trust expiration dates in commercial prep kits?
Are they still good past their due date?

Anonymous Coward said...

Yeah I doubt miniprep kits ever actually expire. The yields may drop but you can always test them to know. I've used antibodies 5 years after their expiration...

muebles en alcorcon said...

This can't have effect in actual fact, that's exactly what I think.

Anonymous said...

It is unnecessary to re-use the spin columns if the price of new spin columns is cheaper enough: $75 for 200 spin columns.