Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Multiple fragment lengths in single 454 titanium run?

    Hi all,

    I have two different fragments that I want to sequence with 454 titanium. One is about 400 bp long and the other about 500 bp long (including gene-specific primers but not fusion primers). Can I run these together in a single run? A lab mate told me that a disproportionately high number of reads will come from the shorter fragment, but it seems a lot of people are doing multiple fragment lengths and getting good results. I have about 100 samples of the 400 bp fragment and about 60 samples of the 500 bp fragment and was hoping to get by with a 1/4 plate run.

    Thanks for any help.

  • #2
    Hi Tom,

    We recommend that amplicons be designed between 200-600 base pairs and within approximately 150 base pairs of each other. Smaller fragments will be preferentially amplified when fragment lengths span outside of these recommendations. With that said, your fragments of 400 bp and 500 bp can be sequenced in a single run.

    APP No. 001-2010 gives some guidance on Amplicon Sequencing using the GS FLX Titanium platform. This Application Brief and additional references can be found on our customer-accessible website at www.454.com/my454.

    Please don’t hesitate to contact your local Roche representative with further queries.

    Kind Regards,

    Nicole
    Technical Support Scientist
    454 Life Sciences, A Roche Company

    Comment


    • #3
      Thanks

      Thank you for your help.

      Comment


      • #4
        When we sequence different sizes of amplicons, we separate them in groups by sizes, emPCR, and then combine DNA beads together for sequencing.

        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
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        9 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