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  • aaron_tao
    replied
    Anyone tried to purify double strand circular DNA by AmPure XP??

    Anyone tried to purify ~1kb double strand circular DNA by AmPure XP?

    Leave a comment:


  • ctguhu
    replied
    good questions, it give me some help, thanks everyone in this question

    Leave a comment:


  • nextgen
    replied
    When you say more effective, you mean the range of the DNA size, distribution or recovery?

    Originally posted by cbird View Post
    We've found SPRI Select is much more effective than Ampure XP for size selection also. Anybody know what the difference is? I seriously doubt it's the beads themselves...

    Check out this paper. http://www.plosone.org/article/info%...l.pone.0010029

    They use varying percentages of PEG and 0.9M NaCl, not 2.5M. They are also using MyOne CA Dynabeads

    Leave a comment:


  • yaximik
    replied
    Originally posted by biozlzyc View Post
    The bead is overdried and there is no DNA in the supernate.What should I do?
    Use TE with 0.05% Tween20 and set it to shake at 37. You may also need to sonicate it a bit to break clumps, but I would not do it if DNA was large

    Leave a comment:


  • cbird
    replied
    We've found SPRI Select is much more effective than Ampure XP for size selection also. Anybody know what the difference is? I seriously doubt it's the beads themselves...

    Check out this paper. http://www.plosone.org/article/info%...l.pone.0010029

    They use varying percentages of PEG and 0.9M NaCl, not 2.5M. They are also using MyOne CA Dynabeads

    Leave a comment:


  • biozlzyc
    replied
    The bead is overdried and there is no DNA in the supernate.What should I do?

    Leave a comment:


  • aperera
    replied
    As mentioned earlier with Ampure XP, we were seeing inconsistent results. Our recent testing with SPRIselect has been going well and the data so far looks amazing! What seem to work best for us is 0.8X and 0.61 double SPRI. Peak at 300 +/- 100bp.

    Leave a comment:


  • aperera
    replied
    I heard there should be less lot to lot variation that you sometimes see when doing size selection with Ampure XP. So in other words, sounds like there is more QC that goes on for SPRISelect. Not sure if there are differences in buffer etc...

    Leave a comment:


  • jtackney
    replied
    Anyone have any experience/thoughts on the new Agencourt product SPRISelect? Is it just higher quality beads of Ampure XP or is something new in the buffer to help with size specificity? All I see is a $50 more expensive product that is already over priced, but I digress....

    Leave a comment:


  • TonyBrooks
    replied
    Originally posted by junorose View Post
    i uesed 25pmol primer,both universal and index primer, in theory, it should be plenty enough; last time i did this experiment, i first added 50ul water to 50ul pcr reaction, then perfromed Ampure XP clean up. i got as much as 55ng/ul *17ul DNA.
    15 cycles is still a lot. We have over amplified with less cycles and similar amounts of primer. It would depend on how much of the PCR input is competent. Have you run the samples on the Bioanalyser? Do you see an over amplification bubble?

    Leave a comment:


  • junorose
    replied
    Originally posted by TonyBrooks View Post
    I think it more likely that you've exhausted your primers during your PCR rather than bead saturation. 15 cycles is a quite a lot of amplification.
    1µL of AmpureXP should contain enough beads to bind a few µg's of nucleic acid.
    i uesed 25pmol primer,both universal and index primer, in theory, it should be plenty enough; last time i did this experiment, i first added 50ul water to 50ul pcr reaction, then perfromed Ampure XP clean up. i got as much as 55ng/ul *17ul DNA.

    Leave a comment:


  • SarahNGS
    replied
    Originally posted by ooriw View Post
    Used AmpureXL on Zymmo purified samples (no salts or detergents), got reasonable yields but the beads did not align well to the tube side but rather settled to the bottom.
    has anyone encountered this- what does it mean?
    What kind of magnet are you using?

    Leave a comment:


  • TonyBrooks
    replied
    Originally posted by junorose View Post
    hi, i am using AmpureXP to perform DNA purification to library chip-seq DNA. i change the size selection ratio from 0.9*-0.2* to 0.8*-0.4*to get a wider size range. one problem i have confront is that no matter how much initiation DNA i use(from 10ng to 160ng), i always get an equal amount of DNA(~30ng/ul*17ul) after PCR amplification(15 cycles). i cannot explain this result, can it be due to DNA capacity of AmpureXP beads?
    I think it more likely that you've exhausted your primers during your PCR rather than bead saturation. 15 cycles is a quite a lot of amplification.
    1µL of AmpureXP should contain enough beads to bind a few µg's of nucleic acid.

    Leave a comment:


  • ooriw
    replied
    Ampure beads don't settle well

    Used AmpureXL on Zymmo purified samples (no salts or detergents), got reasonable yields but the beads did not align well to the tube side but rather settled to the bottom.
    has anyone encountered this- what does it mean?

    Leave a comment:


  • junorose
    replied
    anyone knows the DNA capacity of AmpureXP beads?

    hi, i am using AmpureXP to perform DNA purification to library chip-seq DNA. i change the size selection ratio from 0.9*-0.2* to 0.8*-0.4*to get a wider size range. one problem i have confront is that no matter how much initiation DNA i use(from 10ng to 160ng), i always get an equal amount of DNA(~30ng/ul*17ul) after PCR amplification(15 cycles). i cannot explain this result, can it be due to DNA capacity of AmpureXP beads?
    Last edited by junorose; 12-27-2012, 06:26 PM.

    Leave a comment:

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