Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Longer insert protocol

    Hi all
    For standard DNA preps, we've been happily using the NEBNext kits with Covaris shearing (Agilent protocol) AmpureXP bead size selection (from the NEB protocol). This usually gives us insert sizes of around 2-250bp, which is fine for HiSeq 2x100 sequencing.
    Now we are going to be sequencing a few bacteria strains on the MiSeq with 2x250 reads. Obviously, to make the most of this we're going to need a longer insert size (greater than 400 would probably be OK). Has anyone optimised a gel-free protocol for longer genomic library preps? I was thinking about using the size selection protocol from the 454 rapid libraries (target is 700bp), but the only thing putting me off is the last time I did 454 libraries, I had them approaching 1kb (which would probably be OK on the MiSeq, but I'd prefer inserts to be a bit smaller).
    Thanks

  • #2
    FYI, I tried tweeking the NEB protocol with some success. I now have a library peaking around 390bp (insert size of 260bp).

    a) I sheared to 300bp on Covaris.
    b) Size selected between 0.6X and 0.8X AmpureXP
    c) Increased PCR extension time to 45 seconds (probably not necessary)

    The library is definitely bigger, but I may try a 0.5X-0.6X Size selection next time to minimise the fragments below 300bp and tighten the spread.
    Attached Files

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Essential Discoveries and Tools in Epitranscriptomics
      by seqadmin




      The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
      04-22-2024, 07:01 AM
    • 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

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, Today, 11:49 AM
    0 responses
    13 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, Yesterday, 08:47 AM
    0 responses
    16 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-11-2024, 12:08 PM
    0 responses
    61 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    60 views
    0 likes
    Last Post seqadmin  
    Working...
    X