Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Pre-Hybridization PCR not running efficiently

    Hello all,
    I am working on building my libraries for an NGS project. I am running into a problem in which my pre-amplified libraries have concentrations ranging from 8 ng/uL up to 15 ng/uL. However, when I attempt to amplify these libraries using a Phusion master mix and the primers that are compatible with our adapters for a 12 cycle PCR, the product qubits much lower (.5 ng/uL - 1 ng/uL) and appears to have not amplified. Running a normal QC PCR however I see product on my PCR gel indicating that the adapters ligated properly and are working with the primers. If anyone has any idea what my be causing this or possible troubleshooting steps that would be much appreciated!
    Molly

  • #2
    What type of libraries are you generating? How are you measuring concentrations?

    I would suggest to avoid Phusion for complex libraries. It is fine for amplicon sequencing. Phusion has been shown to introduce comparatively strong biases according to GC content and fragment lengths. Usually, NEB Q5 or Kapa Hifi are used instead.

    12 PCR cycles seems way too much, when starting out with 15 ng/ul. It could be possible that the libraries are overamplified, and thus are mostly single-stranded leading to low measurements.

    Comment


    • #3
      Hi Luc,
      Thank you for your reply. I have also tried to use Kapa Hifi and have seen the same results. I am using a qubit to measure concentrations. While 12 cycles may be to much for the samples that are 15 ng/uL I am observing the same results in libraries that are closer to 1 ng/uL as well. I will try to run my samples for less cycles and see if there is a noticeable difference. Thank you,
      Molly

      Comment

      Latest Articles

      Collapse

      • 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
      • seqadmin
        The Impact of AI in Genomic Medicine
        by seqadmin



        Artificial intelligence (AI) has evolved from a futuristic vision to a mainstream technology, highlighted by the introduction of tools like OpenAI's ChatGPT and Google's Gemini. In recent years, AI has become increasingly integrated into the field of genomics. This integration has enabled new scientific discoveries while simultaneously raising important ethical questions1. Interviews with two researchers at the center of this intersection provide insightful perspectives into...
        02-26-2024, 02:07 PM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 03-14-2024, 06:13 AM
      0 responses
      32 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-08-2024, 08:03 AM
      0 responses
      71 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-07-2024, 08:13 AM
      0 responses
      80 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-06-2024, 09:51 AM
      0 responses
      68 views
      0 likes
      Last Post seqadmin  
      Working...
      X