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  • yaximik
    replied
    Originally posted by katsigner View Post
    Can the beads be reused?? Any idea?
    Sure, why not - if you can trust them in regard to contamination. I guess a very aggressive nuclease can be used for cleaning, like benzonase or cyanase, but can you trust them then in regard to DNA integrity?
    A cheaper alternative could be carboxyl SeraMag from Fisher dispersed in PEG/NaCl solution, but you will have to titer mixing ratios vs precipitation range. Several papers online can give guidelines. In my experience 25% PEG4000/2.5M NaCl works at DNA:beads ratio of as low as 1:0.5.

    Leave a comment:


  • Isequencestuff
    replied
    Originally posted by edawad View Post
    When using Ampure XP beads, the included protocol says to dry for <5 min after the EtOH wash to avoid beads drying out too much and cracking. However most online protocols I see, plus Illumina's Truseq protocol (which uses Ampure XP) all say to dry for 15+ min until the beads crack. Anyone have any experience what difference this makes?

    Also most protocols say to use fresh 70% ethanol to wash, but the Truseq calls for 80%. My understanding is that the higher ethanol concentration might be less efficient at washing away smaller molecules. Has anyone played around with this?

    thanks
    We've had experiences with both: completely dry cracking beads, and also still moist. We can't seem to see a difference in yield between the two using AMPure XP (we haven't used regular AMPure). We use 70% EtOH for Ion Torrent library prep PGM workflow, and 80% EtOH for Illumina library prep Hiseq workflow just because it's in their protocol. We've adapted the Ion Torrent methodology on the Illumina purification protocol: Following aspiration of the last EtOH wash on the magnet, we spin the tubes down and then place back on the magnet. Then suck up the residual EtOH. This allows the beads to dry faster. If you stick to the Illumina Tru-seq protocol you'll have to wait 15+ minutes for the beads to dry (or at least for the residual beads to evaporate. I'm not sure about the EtOH 70/80% differences, I'm rather curious myself. (maybe mean bp size for Sheared DNA input?)

    Leave a comment:


  • Susanne
    replied
    Re. size of Ampure beads, I've also been wondering and reading a bit, and by looking through the Hawkins 1998 patent, I stumbled upon Biomag particles (formerly from PerSeptive, that had been acquired by PE, that became ABI a.f.a.i.k.).
    I understood from searching around that they have a mean diameter of ~1.5 µm and are of irregular shape, thereby having a higher surface area than spherical beads. (However, there are varieties of Biomag particles that can be larger.)
    Hope this isn't too far away from the real size of AMPure beads (I would assume Backman buys the particles somewhere).

    Leave a comment:


  • SarahNGS
    replied
    re-using AMPure beads

    Originally posted by yorkzhou View Post
    Theoretically, there in no reason that the beads can not be reused.

    Beckman Coulter can't be happy about this idea. At least they may argue that you need to decontaminate the beads before binding other samples.

    The homebrewed buffer seems to be promising; maybe someone can do some tests.
    There was a paper published this year by the Broad institute, and they re-use the SPRI beads.

    Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready li …


    BTW I advise anyone doing NGS to find and read all method papers from the Broad institute (Chad Nusbaum), they are the pioneer in NGS techniques and produce really nice & informative publications in (usually) public domain journals.

    Leave a comment:


  • edawad
    replied
    I've just been drying 5 minutes under a hood, spinning down, and resuspending. Ampure XP has been so efficient regardless that I always have plenty of library (usually 10x more than doing the 2nd gel sizeselection, and just as effective at removing the adapter dimers).

    Leave a comment:


  • Gina_P
    replied
    Originally posted by chronicle View Post
    Has anyone tried this out yet or had any thoughts? I'm getting ready to start using Illumina's Trueseq kit as well, and this struck me as a little odd too.
    I use 80% ethanol, and also, I have let my beads dry to the point of cracking (15 minutes) and have seen no ill results from that. I always have gobs of library for sequencing. However, it's good to know that I can also let it dry for a shorter amount of time. Now all those Ampure XP cleanups will go a lot more quickly.

    Leave a comment:


  • cascoamarillo
    replied
    Hi,
    This question is related to AMPure XP but comparing it with the RNAClean XP. In theory the have different features:

    As a leader in R&D genomics services, GENEWIZ provides superior data and high-quality constructs for next generation sequencing, gene synthesis, and sanger sequencing.

    As a leader in R&D genomics services, GENEWIZ provides superior data and high-quality constructs for next generation sequencing, gene synthesis, and sanger sequencing.


    but some people told me that they are the same but with different buffer. Any idea or experience on this issue.

    Leave a comment:


  • Heisman
    replied
    Originally posted by Yevaud View Post
    In my case the drying time is making only resuspension time a little bit longer and usually to be really sure that it's dry I dry beads 30 min under vacuum and nothing is happening to the product. But I have to note that mostly I'm working with the PCR products so maybe there are more stable.
    If it works then great, no worries. A lot of people state that if they dry to the point of cracking then they lose yield, and I think the official protocol states this now as well. But if it works, no need to change.

    Leave a comment:


  • Yevaud
    replied
    In my case the drying time is making only resuspension time a little bit longer and usually to be really sure that it's dry I dry beads 30 min under vacuum and nothing is happening to the product. But I have to note that mostly I'm working with the PCR products so maybe there are more stable.

    Leave a comment:


  • Heisman
    replied
    Originally posted by chronicle View Post
    Awesome! Thanks for the prompt reply! Hopefully I will have similarly good results.

    Woops - should have noted - do you use a short dry time (<5 min like the Ampure XP protocol calls for), or do you go for a longer drying time (i.e. 15 min) as specified in the Illumina protocol? It seems like for such a small volume, this is a fairly excessive dry time, but I'm not sure how it will affect the Ampure Beads.
    I go for as short as possible that will allow me to feel confident it's dry. If I'm working with only a couple of tubes, I will suck out drops with a pipette to make it faster, haha.

    Leave a comment:


  • chronicle
    replied
    Originally posted by Heisman View Post
    I've done a bunch of bead purifications. I see pretty good results either way in terms of drying time. One thing to keep in mind is that if you use 70 or 80% ethanol, the ethanol will evaporate first and the last liquid you see will be water. So it can be a tiny bit wet and you can feel pretty confident that you have gotten rid of all of the ethanol. No idea about which ethanol percent to use.
    Awesome! Thanks for the prompt reply! Hopefully I will have similarly good results.

    Woops - should have noted - do you use a short dry time (<5 min like the Ampure XP protocol calls for), or do you go for a longer drying time (i.e. 15 min) as specified in the Illumina protocol? It seems like for such a small volume, this is a fairly excessive dry time, but I'm not sure how it will affect the Ampure Beads.
    Last edited by chronicle; 10-07-2011, 04:21 PM.

    Leave a comment:


  • Heisman
    replied
    Originally posted by chronicle View Post
    Has anyone tried this out yet or had any thoughts? I'm getting ready to start using Illumina's Trueseq kit as well, and this struck me as a little odd too.
    I've done a bunch of bead purifications. I see pretty good results either way in terms of drying time. One thing to keep in mind is that if you use 70 or 80% ethanol, the ethanol will evaporate first and the last liquid you see will be water. So it can be a tiny bit wet and you can feel pretty confident that you have gotten rid of all of the ethanol. No idea about which ethanol percent to use.

    Leave a comment:


  • chronicle
    replied
    Originally posted by edawad View Post
    When using Ampure XP beads, the included protocol says to dry for <5 min after the EtOH wash to avoid beads drying out too much and cracking. However most online protocols I see, plus Illumina's Truseq protocol (which uses Ampure XP) all say to dry for 15+ min until the beads crack. Anyone have any experience what difference this makes?

    Also most protocols say to use fresh 70% ethanol to wash, but the Truseq calls for 80%. My understanding is that the higher ethanol concentration might be less efficient at washing away smaller molecules. Has anyone played around with this?

    thanks
    Has anyone tried this out yet or had any thoughts? I'm getting ready to start using Illumina's Trueseq kit as well, and this struck me as a little odd too.

    Leave a comment:


  • yorkzhou
    replied
    Originally posted by katsigner View Post
    Can the beads be reused?? Any idea?
    Theoretically, there in no reason that the beads can not be reused.

    Beckman Coulter can't be happy about this idea. At least they may argue that you need to decontaminate the beads before binding other samples.

    The homebrewed buffer seems to be promising; maybe someone can do some tests.

    Leave a comment:


  • niceday
    replied
    if the beads dry to cracking it can be difficult to elute the DNA in the time mentioned in the protocol.

    Some people make up 70% by eye and because 7ml of EtOH and 3ml of water has a volume less than 10ml they make it up to less than 70%.

    Leave a comment:

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