Unconfigured Ad

Collapse
X
 
  • Filter
  • Time
  • Show
Clear All
new posts
  • Synthetica
    Junior Member
    • Dec 2013
    • 4

    Nextera XT library preparation

    I am using Nextera XT for library preparation.
    The kit suggests 12 cycles of PCR after tagmentation.
    If I run 5 ul of PCR product after 12 cycles I do not see any smear on my 1.5% agarose gel. If I run 13 cycles then I see the smear.
    I am wondering if this is normal or if I should see a smear after 12 cycles.
    I will try to go to the Bioanalyzer to see my library but I am not sure it will give me a better answer.
    Any help or comment could be really useful.
  • nucacidhunter
    Jafar Jabbari
    • Jan 2013
    • 1250

    #2
    I do not run my libraries on gel to check, so I do not know if one should see smear or not. If you see smear after 13 cycles by running 5 ul of PCR, then using 10 ul after 12 cycles should give similar results.

    Comment

    • smoics
      Member
      • Jan 2016
      • 15

      #3
      This is normal. Many people modify the protocol to increase the number of PCR cycles like this.

      Comment

      Latest Articles

      Collapse

      • SEQadmin2
        Advanced Sequencing Platforms Tackle Neuroscience’s Toughest Genomics Problems
        by SEQadmin2



        Genomics studies in neuroscience face a special challenge due to the brain’s complexity and scarcity of samples. Mapping changes in cell type and state using conventional next-generation sequencing methods remains challenging. Advances in technologies like single-cell sequencing, spatial transcriptomics, and long-read sequencing have opened the door to deeper studies of the brain and diseases like Alzheimer’s, amyotrophic lateral sclerosis (ALS), and schizophrenia.
        ...
        Yesterday, 11:10 AM
      • SEQadmin2
        Cancer Drug Resistance: The Lingering Barrier to Rising Survival
        by SEQadmin2



        Cancer survival rates have significantly increased in the last few decades in the United States, reaching a combined 70% 5-year survival rate by 2021. Behind this number, there are years of research to find new therapies, drug targets, and early detection methods. But there is one core challenge that keeps slowing down these advances, and it’s about drug resistance.

        There is no single reason why many patients don’t respond to treatment as expected. Cancer is...
        07-08-2026, 05:17 AM
      • GATTACAT
        Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing
        by GATTACAT
        Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
        07-01-2026, 11:43 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by SEQadmin2, Yesterday, 10:04 AM
      0 responses
      10 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, 07-08-2026, 10:08 AM
      0 responses
      9 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, 07-07-2026, 11:05 AM
      0 responses
      16 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, 07-02-2026, 11:08 AM
      0 responses
      31 views
      0 reactions
      Last Post SEQadmin2  
      Working...