A penny for your method: PCR cleanup

Very rarely, in scientific research, do we get the opportunity to do less with more. As a result, one thing that we are good at is stretching a dollar. To (hopefully) contribute something back to the struggle, I’ve decided to start an intermittent set of posts that focus on reducing the cost of common laboratory methods.

Sometimes, I’ll present protocols or links to protocol, and others I will likely lay out many of the essentials, and let you fill in the blanks. Occasionally, my suggestions will be pretty simple – but saving money is saving money, right?

As part of this effort, please send the real golden-nugget, dollar-saving lab hacks my way (molecularecologist+pennymethod@gmail.com), and I’ll do my best to curate what I get and post the golden-est of nuggets I receive right here (with attribution, of course).

Now, with that out of the way, I’ll start the party with PCR cleanup – specifically medium-throughput cleanup (i.e., 96- or 384-well).

The gist of PCR cleanup is that you often want to remove unincorporated primers, left-over dNTPs, salts, and other “stuff” from amplicons before you do other things with them. Kits are a handy solution to the cleanup “problem” because they’re pretty fast, extremely convenient, and largely idiot-proof. Commercial options for these sorts of kits include those from Qiagen and Zymo, among others. The Qiagen QIAQuick™ kits (p/n 28181) list at $670.00 per kit (4×96-well plates) or $1.75/sample. The Zymo ZR96 DNA Clean & Concentrator™ (p/n D4023) kits (2×96-well plates) list at $189.00 per kit or $0.98/sample.

But, all of these are too rich for my blood. I could probably do EtOH precipitation in 96-well plates, if I were really hard-core, but I’m not always really hard core nor do I like to spin plates eternally, if i can help it. And, 96-well EtOH precips in inexperienced hands can go horribly wrong. Bottom line is that there’s a better solution available – easy and idiot-proof like the commercial kits and inexpensive like EtOH precipitation:

Cost for this operation is $0.25/sample for the DNA Filter plate, $0.05/sample for the flow-through plate, and $0.04/sample for the DNA storage plate – for a grand total of $0.34/sample (chemical cost is largely negligible on a per sample basis, but add $0.01/sample, if you like). This represents a savings of 65% over the Zymo kits and 80% over the Qiagen kits. You can reduce costs further by re-using the flow-through plates – treat them in 10% bleach if you are concerned about cross-contamination.

Enjoy.

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About Brant Faircloth

I'm an Assistant Researcher at the University of California - Los Angeles. My interests include mating behavior, social behavior, the (immuno-)genetic basis of mate choice, genomics of non-model organisms, metagenomics, computer programming, and the integration of molecular and field biology.
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  • Dilara Ally

    If only I had known this for my last project. We sequenced 288 genomes of a ssDNA virus (ID11). But in order to sequence the ~6kb genome, we had to PCR the genome into 2 halves. I spent a lot of time doing single tube PCR clean ups. Ugh!

  • I’m a big fan of the magnetic Agencourt Ampure XP beads. If you buy the 60 ml bottle of the it works out to be about $1.60 per 50 ul PCR rxn or $0.80 for a 25 ul PCR rxn. You also need a magnet stand. I recommend the Agencourt SPRIPlate Super Magnet Plate which costs ~ $200. Of course the more beads you buy the cheaper the cleanups. Also, you get really high and consistant recoveries. Every sample I prep for NextGen sequencing gets a final XP bead cleanup. Just be sure to make your EtOH fresh each time. Oh, and it’s super nice not to have to deal with a plate centrifuge; you can do the whole process right on your bench top.

    • Kelly Barr

      I just checked, and it looks like that Super Magnet plate is now running upwards of $850. Inflation?

  • I’ll second the SPRI/AMPure cleanup – but I was going for ultra-cheap here. In fact, I have an ultra-cheaper method I’ve been debugging that cuts the costs to basically that of the plates you need for the cleanup and your time.

    On the subject of SPRI, it is entirely awesome and you are spot on that recovery is much, much better than any cleanup “kit” using column purification. In fact, I’ve been using AMPure for a few weeks on another project and it performed heads and tails above column based approaches.

  • Chokchai

    I just stumbled on your post and would like to ask you for some advice. I have been using three different PCR clean-up kits (including QIAGEN) to directly purify PCR products I got from colony PCR of different Actinomycete bacteria. The yields were very low although I had done the same thing with other bacteria and was successful in getting a good amount of DNA. Would you happen to have any idea what could be going wrong in my case with the fool-proof PCR clean-up kits? Also, have you ever tried using EtOH precipitation with PCR products for subsequent sequencing? All sequencing providers suggest the PCR amplicons should be purified with the kits but I am wondering how bad it would be with EtOH precipitation. Thank you for your help.

    • Hi Chokchai,

      I’m obviously slow in responding, but in case you are still wondering… for your first question – I’m not sure what would be causing problems. I might check the dates on your reagents to see if they are still good – otherwise, I’m really not sure what could be causing issues.

      To address your second question – for PCR amplicons prior to Sanger sequencing, I generally use a homebrew ExoAP mix which should be detailed in the following link:

      http://dna.uga.edu/docs/Isolating%20microsatellites%20DNA%20loci.pdf

      I have not used EtOH precip for amplicons prior to sequencing (although I have used it to clean up sequencing reactions).

  • Marcel Marceau

    Mr Brant Faircloth,

    Have you posted the protocol yet? The post above shows a list of consumables and their cost, but I do not see the protocol per se. Could yo email me the protocol?

    Tank you,

    Marcel

  • Hi Marcel,

    The protocol is in the linked document at the Whatman site:

    http://www.whatman.com/UserFiles/File/Protocols/Bioscience/384%20well%20PCR%20Clean-up%20Protocol%20for%20Centrifuge.pdf

    cheers,
    b

  • Marcel Marceau

    Thank you very much for your answer.

    Happy 2013!!

  • HK

    Thank you for the useful post!

  • Joe Magro

    A great method that I have not seen mentioned yet is ExoSAP-IT (Affymetrix). It is a simple enzymatic cleanup that is less expensive than columns and without the downsides of loss of product you have with columns and beads. Great sequencing results, too.

  • Brian Tetkowski

    Yes, ExoSAP-IT Cleanup Reagent is an excellent option. 100% recovery and they also have a High Throughput formulation HT-ExoSAP-IT. Best cleanup product on the market and a great value! 1 step, 1 tube and fast.