Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • loss of library DNA in the last purification, need help

    Hi,
    I'm doing library preparation for SOLiD 5500. I have no problem during the process (size selection, dA tail, adaptor ligation etc), until the purification step after amplifying the library. We have no DNA left after purification . I used Agencourt AMPure beads for purification. Is anybody has the same problem and know is/are the cause(s), and know how to solve it? Our group has the same problem before.
    Many thanks

  • #2
    Hi, Anjani,
    i do not know what kind of your library, i try to contribute some minds.Ok, Agencourt AMPure beads is excellent product for purefication for DNA and RNA, they have RNA Clean beads for clean RNA specificlly. even i already try single product, it is work very good.
    i just gess they use PEG8000 and NaCl as bead suspention buffer. maybe the concentration is 13%PEG/1.25M NaCI/10mM MgCI2, we dont care that. if you dont like that, try these way:62% ethanol in final concentration, plus 500mM NH4OAc in final concentration. for example: if your DNA/RNA solution is 100ul, add 100% Ethanol 221ul and 36ul 5M NH4OAc to total 357ul. on magnetic rack 1min, discard original bead supernatant, suspend in your made solution. at room temp mix well and incubate less 5minutes, then set in a magnetic rack, discard supernatant, dry to near crack pellet, dont over dry!! elute your H2O or TE.
    remember please, 55%-62% ethanol will give your gold range for purefication process, depending your experiment request.
    good luck

    Comment


    • #3
      This has also happened to me once during the final size selection of the library end product. I must have not been careful with getting ever microliter right and I had no library of the expected size in the end. So I just repeated the washing and cut off on the same beads being extra careful (hoping my product was still sticking to them) and managed to successfully elute the product from the beads.

      Comment


      • #4
        We had the same problem several time, we stopped using beads, now using spin columns and we have no problem

        Comment

        Latest Articles

        Collapse

        • seqadmin
          Strategies for Sequencing Challenging Samples
          by seqadmin


          Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
          03-22-2024, 06:39 AM
        • seqadmin
          Techniques and Challenges in Conservation Genomics
          by seqadmin



          The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

          Avian Conservation
          Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
          03-08-2024, 10:41 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, Yesterday, 06:37 PM
        0 responses
        11 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        51 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        67 views
        0 likes
        Last Post seqadmin  
        Working...
        X