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  • #16
    Does anybody know what the composition of the elution buffer is? Shouldn't that be almost water?

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    • #17
      Water will work. Typically I use 10mM TrisHCl pH 8.5.

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      • #18
        Ampure XP size

        Anybody know how big the Agencourt Ampure XP magnetic beads are?

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        • #19
          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

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          • #20
            Can the beads be reused?? Any idea?

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            • #21
              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%.

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              • #22
                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.

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                • #23
                  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.

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                  • #24
                    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.

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                    • #25
                      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.

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                      • #26
                        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.

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                        • #27
                          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.

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                          • #28
                            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.

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                            • #29
                              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.

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                              • #30
                                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.
                                - Gina

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