Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • TruSeq PCR free DNA with 16 amplicons

    New member and new to running my own MiSeq Sequencer!

    We are trying to figure out how to modify TruSeq PCR free DNA library prep kit protocol to make a library with our pool of barcoded amplicons (16s and ITS) for sequencing on the MiSeq. Has anyone else done this or seen any published protocols combining these methods?

    The problem I am running into is that we use SequalPrep Plates to normalized our PCR amplicons (~350bp) and then pool. This theoretically provides us with 1.9ml of pooled PCR product at ~1ng/ul (we ended up with about 0.7 bc of negtive controls and failed PCR reactions).

    We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).

    Speed vac to concentrate would be problematic due to the elution buffer used in SequalPrep. I tried a Zymo clean and concentrator column, but lost 60% of the yield. SequalPrep does have a sequential elution recommendation, which I have not tried yet, but at 5 min per well it will be time limiting. Any suggestions?

  • #2
    This seems to be the really expensive way to add index and adapters. Why not just make the big indexed primers (a la Kozich or Caporaso) or 2 step PCR?
    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
      Originally posted by thermophile View Post
      This seems to be the really expensive way to add index and adapters. Why not just make the big indexed primers (a la Kozich or Caporaso) or 2 step PCR?
      It sounds like maddo1ck has already indexed and pooled, now just needs to add the Illumina bits to the end, i.e. just a single ligation reaction.

      maddo1ck, try a Amicon centrifugal ultra filtration concentrators . They are simple to use and recovery is generally excellent.

      Comment


      • #4
        The idea was that since we are a fairly small lab and don't have hundreds of samples for sequencing this method would be flexible in allowing us to do both PCR amplicons and WGS for individual samples if we wanted.

        Thanks for the suggestion kmcarr, I will look into those. I also realized we could do a serial dilution with sequal prep to reduce our volumes so we are going to try that first to avoid any potential loss thru a spin step.

        Comment


        • #5
          Hi Maddoc1ck - we found this protocol helpful when using a 'home-made' PCR free approach. http://www.ncbi.nlm.nih.gov/pubmed/21431776

          Good luck with your workflows.

          Comment


          • #6
            Originally posted by maddo1ck View Post

            ...

            We would start the TruSeq protocol with the dAtailing step, which calls for 15ul of sample that I guess is around 800ng since you start the full protocol with 1100 (for a 350bp insert).
            ...
            Definitely start with the end repair; it adds a phosphate group that probably is necessary!

            We usually add about 100 ng of PCR product, and we get around 50% of these fragments with adapters successfully ligated at both ends, which is more than enough.

            We have moved to the two-step PCR protocol, it has many advantages.

            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
            10 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            9 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