Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • PCR amplicons primer design - need advice

    Hello,
    I have a list of dna sequences corresponding to a family of HSP proteins in Bacteria (800 seqs of length average 570nt). I would like to design PCR primers for miseq amplification (2*250pb).

    The insert size should be around 460bp since this is the length that is good quality from the miseq sequencing to be analysed.

    I was wondering if you can suggest a pipeline and tools to design my PCR primers that cover most possible species, which are likely to be degenerated.

    My first idea to create an alignment for all sequences using Muscle.

    From there how do i get degenerated primers ?

    Thanks for your comments.
    Last edited by danova; 10-26-2016, 11:14 AM.

  • #2
    are you looking for snp's? if so you should be designing your primers to hit a smaller target because you want a lot of overlap in your sequences. Last time I designed primers (it was a while ago) I used ARB-aligned my seqs and used the primer design tool to get potential targets.

    The above is assuming that you've checked lit for other primers hitting your target gene, compiled a list of those, tested in silico against your seqs, and found none appropriate.
    Last edited by thermophile; 10-27-2016, 05:35 AM.
    Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

    Comment


    • #3
      Not looking for SNP , just the HSp proteins that are very conserved across species.

      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, Yesterday, 06:37 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X