Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • primer dimer

    Hi everyone,
    We have to do a 454 GS junior run but amplimers coming up from our two steps PCR have a visible primer dimer. Do you think this will influence the quality of the run (more short reads, etc) or the AMpure purification is sufficient to get rid of all dimers?
    Thank you.

  • #2
    You may need to do multiple ampure clean-ups to get rid of the dimers. (we occasionally do 2 ampure and a gel slice purification).

    Comment


    • #3
      Hi jdelton,

      Do you mind sharing what's your typical recovery from ampure and gel purification?

      When we follow Roche's clean up protocol our recovery is super below like under 10% (even <1%). It becomes not practical to perform clean up when you almost end up with nothing.

      Thanks!

      Comment


      • #4
        We actually modified Roche's clean up for our ampure. We do 70% of the volume (more or less depending on the ampure calibration which we do with out sizing solution) of the pool - Pool=100ul ampure = 70ul. This clean up is eluted in 100ul and started again. Our final elution is in 30ul. Overall I couldn't tell you what our percent recovery is, but it's enough that it is clearly able to be visualized on an agarose gel and using a bioanalyzer. And more than enough to dilute into emPCR.

        Comment


        • #5
          Originally posted by jdelton View Post
          We actually modified Roche's clean up for our ampure. We do 70% of the volume (more or less depending on the ampure calibration which we do with out sizing solution) of the pool - Pool=100ul ampure = 70ul. This clean up is eluted in 100ul and started again. Our final elution is in 30ul. Overall I couldn't tell you what our percent recovery is, but it's enough that it is clearly able to be visualized on an agarose gel and using a bioanalyzer. And more than enough to dilute into emPCR.
          Do you omit the Sizing Sizing solution completely when performing the AMpure clean up? I have had mixed results using the Sizing Solution, and often wondered if I'd be better off without it.

          Comment


          • #6
            Yes we omitted it completely. It was a pain, expensive, and the whole purpose was to have consistent results, yet you still have to calibrate it every time. Plus it didn't flow into our workflow easily.

            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, 03-27-2024, 06:37 PM
            0 responses
            13 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-27-2024, 06:07 PM
            0 responses
            11 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-22-2024, 10:03 AM
            0 responses
            53 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-21-2024, 07:32 AM
            0 responses
            69 views
            0 likes
            Last Post seqadmin  
            Working...
            X