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What AMPure is not good for

October 26, 2012

So I’ve been pretty excited about AMPure since I learned you could order the beads without the PEG for a fraction of the cost and I’ve been eliminating QIAquick and phenol-chloroform extractions/ETHANol precipitations from my molecular biology vocabulary.  But it appears there is a big limitation to AMPure.  With QIAquick and phenol-extractions, the proteins are denatured then purified away by chromatography or organic partitioning respectively.  Qiagen Buffer PB has 5M guanidine hydrochloride.  Whereas with AMPure the proteins are only purified away by not precipitating on the beads (AMPure buffer is 1.25 M NaCl and PEG).  So in some cases where trace enzymatic activity carryover can be an issue, AMPure may be problematic.

I found such a case.  I was making the monomers for ICA TALE assembly and used AMPure.  The assembly failed.  What this entailed was PCR amplifying a 100 bp DNA fragment, cutting it with BsmBI, which cuts at the ends, and ligating the fragments together.  If you look at the gel below you can see the monomers (100 bp DNA fragments) PCR amplified and then cut with BsmBI.  The ones purified with QIAquick look nice and crisp but the one purified with AMPure look a little degraded.  What I think is happening is some carryover polymerase from the PCR reaction is trying to blunt the ends.  A couple days work and a significant amount of reagents down the drain but a good lesson learned.  Thought I’d try to save some other from the same mistake.

Now the question is, if you make AMPure beads with guanidine hydrochloride, will it function like QIAquick?  I gave it a quick little go and the problem is getting everything into solution, but I think initial data looks promising.  How long until some company patents it?

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15 Comments
  1. mc1061dm1 permalink

    Just a reminder, PEG may inhibit enzyme activity, which is an old folklore passed from biochemist of last generation.

    • ethanomics permalink

      I guess the thing to keep in mind is no purification method is perfect and it’s important to know the deficiencies in whatever method you are using. But yeah, I bet you are right that there is some left over PEG in AMPure purification that could inhibit some enzymes.

  2. mc1061dm1 permalink

    How much DNA do you need for NGS? Our facility asked me to submit 10 nmol each index in 10 ul, which is very high, is it?

    • ethanomics permalink

      This is a note from the facility (EMBL Genecore) that has been doing our samples lately.

      Hey Ethan, the standard illumina supported protocol assumes 2 ul of 10nM library. If we bend the rules a little, the lowest possible molarity would be 1.1nM and we would need 18ul of that..however I wouldn’t go that low.

      2 μl of 10 nM library is 20 fmols, so there must be some sort of miscommunication going on. Generally I like to have enough so I can see the library when I run it on the Bioanalyzer.

  3. mc1061dm1 permalink

    I guess the thing to keep in mind is no purification method is perfect and it’s important to know the deficiencies in whatever method you are using.

    You are right, understand a protocol in and out is important, and I also believe that leftover enzyme will have a stronger effect (chew the end of DNA) than) PEG.

  4. mc1061dm1 permalink

    so there must be some sort of miscommunication going on.

    You are right again, I called them, and told 10 mM not 10 mmol (as a Chinese, it’s not easy for me to distinguish the two). Problem solved.

  5. mc1061dm1 permalink

    I have amplified my library, and will do last purification from PCR product. Usually Ampure is suggested for this step, while I was wondering whether Qiagen or Zymo PCR purification kit is also OK. In the old protocol (published 2012, developed 3-4 years ago), Qiagen kit was used, while suddenly, everyone is using Ampure. Is there any reason why Ampure kicked Qiagen out?

    Thanks

    Ming

  6. ethanomics permalink

    Hi Ming, You can use the Qiagen columns for every step except the last, although I think the recovery is better from AMPure. The Qiagen columns are not very effective at getting rid of unused primer and the left over PCR primers will block your library from binding to the flow cell. It’s explained here:
    http://www.ncbi.nlm.nih.gov/pubmed/19034268

    I think a big reason protocols switched to AMPure was that it can all be done with a robot or a multichannel pipet. I don’t use either but I think the recovery may be a little better with the AMPure beads.

    Ethan

  7. mc1061dm1 permalink

    Happy new year!

  8. mc1061dm1 permalink

    Just let you know, I talked with IDT guy about the oligos for adaptors recently, and they told me something interesting: the oligos might crosstalk/see each other/contaminant during manufacturing, i.e. in index 1 oligo, some index 2 or 3 might exist.

    To overcome this, they developed a process called “TruGrade” to minimize the contamination.

    Of course, they told me the potential problem because they wanted to sell me the “TruGrade” oligos, and I bought some.

    Whatever, the potential contamination of other index adaptors are scary, it’s something I didn’t know before.

    • Wow, that’s something I never thought about. I always assumed the oligos I was getting from IDT were not contaminated. But I guess they’re being synthesized on a machine that is used to synthesize other oligos. It never made sense to me to get HPLC or PAGE purification but cross contamination with other barcodes is not an issue I want to deal with if possible. Thanks for the heads up. And Happy new year!!

  9. thucnghi permalink

    Can’t you just add Qiagen PB buffer after your PCR reaction, and then use AMPure and purify as normal? Qiagen PB contains guanidine hydrochloride. This was suggested to me in a protocol, and I have use it, and the purification works fine.

    By the way, I like your blog! I am starting to do RNA-seq and I appreciate your thoughts on some of the things I think about.

  10. mriviere permalink

    hi thucnghi- could you describe that protocol, tell me it’s location?

    • thucnghi permalink

      The protocol is not published, so I can’t point you to it. But what I did was add equal volume of PB to the sample, then add equal volume (or more) of Ampure beads to the new mixture. Example: 25 ul of sample + 25 ul of PB, then add 50-60 ul of Ampure beads. I don’t have a problem with enzymes carrying over to the next reaction, so I have been omitting the PB in my clean up. That means that I am not sure if PB made a difference in my case, and not sure if it will in yours. But you can give it a try!

  11. mriviere permalink

    thanks very much!

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