Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • DNA 2D Barcoding for Mitochondrial DNA

    Hello everyone! My name is Kramer Kaplan, and I am a current graduate student working on fish genetics. My advisor and I are learning how to go through the process of 2D sequencing to obtain the entire mitochondrial genome of two species of freshwater stingrays. I wanted to submit a post here with several questions that I had. We have been using a PCR Barcoding Kit (EXP-PBC001) that has the twelve barcodes and barcode adapter mixes (BCA). I was wondering when you use that adapter mix because it does not say anything about that in the protocol. I'm following the "PCR barcoding amplicon sequencing for the MinION device using SQK-NSK007." As I have been following this protocol, the issue I have been seeing is my concentrations of DNA keep getting less and less until I have less than 20ng/ul. Before prepping the MinION, I am supposed to have around 150ng/ul of material. My first found PCR product showed about 10kB on a gel, and the concentrations were high enough, so I truly do not know what the issue is. If anyone could shed some light on these issues or offer any advice, that would be greatly appreciated!

  • #2
    Hi! You can ligate the BCA to any DNA and then do a barcoded PCR, e.g if you want to use genomic DNA or have amplicons without the ONT pcr-priming tail. We also tend to get lower than recommended yield of DNA after the final streptavidin bead purification, but I think 20 ng/ul is still high. If you only get 20 ng in total then something is likely wrong, perhaps adaptors are too old or your starting concentration was off.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Recent Advances in Sequencing Analysis Tools
      by seqadmin


      The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...
      Today, 07:48 AM
    • 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

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, Today, 07:17 AM
    0 responses
    11 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 05-02-2024, 08:06 AM
    0 responses
    19 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-30-2024, 12:17 PM
    0 responses
    20 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-29-2024, 10:49 AM
    0 responses
    28 views
    0 likes
    Last Post seqadmin  
    Working...
    X