Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Amplicon sequencing - fusion primers ordering

    Dear all,

    we would like to order fusion primers for 454 GS Junior amplicon sequencing.

    When consulting the manual, we are not sure if the primers need to have Phosphorothioated DNA bases on the ends and if B primer needs to be labeled with biotin (or is this true just for shotgun sequencing)?

    Thank you for your help.

  • #2
    Amplicon sequencing - fusion primers ordering

    Hi,

    The phosphorothioate bases are there to inhibit any exonuclease activity, so you can order them without. It did work in our hands but if you get any contamination you'll have a problem!

    The biotin on the B primer is necessary for the enrichment (enrichment beads are streptavidin coated magnetic beads) of the amplified beads, regardless the type of library.

    Comment


    • #3
      For the FLX, the primers do not need to be biotinylated; standard primers work fine. Biotin doesn't come into the process until the enrichment stage, and the biotin there comes from the enrichment primer, not from the primers you used to create your library. Furthermore, only one molecule of the original library you make with your primers will end up on the bead and even if it was biotinylated, the biotin molecule would be stripped off (along with that original molecule) when you add the melt solution.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Current Approaches to Protein Sequencing
        by seqadmin


        Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
        04-04-2024, 04:25 PM
      • 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

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 04-11-2024, 12:08 PM
      0 responses
      14 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 10:19 PM
      0 responses
      19 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 09:21 AM
      0 responses
      16 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-04-2024, 09:00 AM
      0 responses
      43 views
      0 likes
      Last Post seqadmin  
      Working...
      X