Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Low Molecular Weight sample and primer dimers

    I recently ran a Low Molecular Weight sample on our 454 and got a bad result. Most of the sequences were just primer dimers. Our samples are pooled PCR products, 200-600 bp in length. The LMW protocol says to skip the AMPure size selection/cleanup step after adapter ligation, but this allows primer dimers to be sequenced.
    I want to eliminate the primer dimers, but I am afraid that if I do the AMPure cleanup, I will loose part of my sample DNA.
    Any ideas about how to eliminate the primer dimers for the LMW sample?
    I have attached a plot of the sequence read lengths from the last run.
    Attached Files

  • #2
    Hi, if you don't want to use Ampure clean-up or gel extraction (I'm just assuming this would drop out for the same reasons), did you think about something like using a Centricon (or Microcon)? Millipore claims that they cannot only be used for proteins, but also provides DNA cut-off sizes:
    Centricon-30 ssDNA cot-off: 60 nt, dsDNA cut-off: 50 bp
    I think I have read something like 90% retention. I guess every additional step will not have 100% yield, but is that necessary? If your samples are PCR products, you should have plenty of everything.

    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
    26 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    29 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 09:21 AM
    0 responses
    25 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-04-2024, 09:00 AM
    0 responses
    52 views
    0 likes
    Last Post seqadmin  
    Working...
    X