Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • MiSeq Reagent Kit v3

    Hello!
    Has anyone tried to perform RNA-Seq and ChIP-Seq with the new MiSeq Reagent Kit v3?
    What about the throughput?
    Any feedback?
    Thanks
    Paolo

  • #2
    We just did some stranded RNAseq libraries with MiSeq v3 kits.

    Worked perfectly. 1340 k/mm^2 density and 32M reads (29M passed).

    rgds
    Mads

    Comment


    • #3
      Originally posted by MadsAlbertsen View Post
      We just did some stranded RNAseq libraries with MiSeq v3 kits.

      Worked perfectly. 1340 k/mm^2 density and 32M reads (29M passed).

      rgds
      Mads
      I found 2 protocols on library loading, which differ greatly in load ranges. One starts from 10 nM libraries and provides dilution tables from 20 pM to 50 pM final library concentrations - without giving any hints at least what to start from to get 1200-1400 cluster density. Another manual starts from 4 nM library and provides dilution tables up to 20 pM final library concentration - again without a clue what to use to get to the new higher density. Which is correct manual?
      My libraries are made from fragmented DNA, so I started from 10 nM library and diluted to final 25 pM according to the first protocol. I got only ~930 density. So, the entire range in the second protocol would lead to underutilization of v3 capabilities. Any comments from other experiences?
      Last edited by yaximik; 09-21-2013, 06:10 PM.

      Comment


      • #4
        All right, I did another run with the same library using the first protocol, but loaded the flow cell at ~33 pM instead of 25 pM as in the previous run. I got density up to 1144 with 85.2% PF as compared to density of 928 with PF 88% at 25 pM library load. Looks like the protocol 1 is the way to go.
        Last edited by yaximik; 09-21-2013, 06:13 PM.

        Comment


        • #5
          Originally posted by yaximik View Post
          All right, I did another run with the same library using the first protocol, but loaded the flow cell at ~33 pM instead of 25 pM as in the previous run. I got density up to 1144 with 85.2% PF as compared to density of 928 with PF 88% at 25 pM library load. Looks like the protocol 1 is the way to go.
          Nice to see that you got it to work. We use the new loading reccomendations of 20 pM. However it is very difficult to estimate the right concentration of the samples due to the differences in insert length although we run all samples on a TapeStation. We are usually happy if we hit +/- 20 % of the density we aimed for.

          rgds
          Mads

          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
          7 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          7 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          66 views
          0 likes
          Last Post seqadmin  
          Working...
          X