Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Optimization of the cDNA-PCR sequencing protocol​

    I'm using the cDNA-PCR Sequencing Kit (SQK-PCS111) to sequence full-length transcripts from human brain tissue. I have followed the protocol from Oxford Nanopore Technologies and I have obtained about 10 ng of cDNA per sample. However, when I run the QC step using the Qubit and the TapeStation, I see a very low concentration of cDNA (around 1 ng/ul) and a very broad size distribution (from 200 bp to over 10 kb). How can I optimize the cDNA-PCR sequencing protocol to get higher yield and quality of cDNA?

  • #2
    Most optimisation around PCR sequencing is around optimising the PCR itself.

    If your yield is about 1/10th of what you want, then as a simple initial fix try running the PCR for four more cycles.

    A broad size distribution is typical for cDNA sequencing; we usually get a similar range (i.e. 0.2kb - 10kb), averaging 0.6 - 1.5 kb per read depending on how good the input RNA is. Fresh is best - better, less degraded RNA will produce longer reads, more reads, and a more productive sequencing run overall.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Exploring the Dynamics of the Tumor Microenvironment
      by seqadmin




      The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
      07-08-2024, 03:19 PM
    • seqadmin
      Exploring Human Diversity Through Large-Scale Omics
      by seqadmin


      In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
      06-25-2024, 06:43 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 07-10-2024, 07:30 AM
    0 responses
    29 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-03-2024, 09:45 AM
    0 responses
    201 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-03-2024, 08:54 AM
    0 responses
    212 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-02-2024, 03:00 PM
    0 responses
    193 views
    0 likes
    Last Post seqadmin  
    Working...
    X