Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Is a purification step between cDNA generation and library construction necessary?

    Hi all,
    Recently, I was wondering that is it necessary to perform a purification step after cDNA generation and prior to library construction? Because I am gonna do the cDNA generation in a very tiny volume. The library construction can be either ligation-based or tagmentation-based.
    Best!
    Gary

  • #2
    Originally posted by kobeho24 View Post
    Hi all,
    Recently, I was wondering that is it necessary to perform a purification step after cDNA generation and prior to library construction? Because I am gonna do the cDNA generation in a very tiny volume. The library construction can be either ligation-based or tagmentation-based.
    Best!
    Gary
    it´s not necessary at all! Just dilute the PCR product to reduce the amount of salt, take an aliquot for ligation/tagmentation and go on. I tried it myself and it works quite well. I never compared the efficiency with a "standard" protocol, though. Based on what I see on the Bioanalyzer (haven´t sequenced any sample yet) I would be surprised to find out that the difference is so dramatic.
    good luck!

    Comment


    • #3
      No, you can do end repair after the 2nd strand reaction without purification.

      Comment


      • #4
        I believe the main reason you typically see cleanup between 2nd strand and end repair is that the 2nd strand buffer for most systems has ammonium sulphate in it, and the T4 PNK used in end repair is potently inhibited by ammonium. It might work out if the reaction is diluted enough that the ammonium is a nonfactor.
        Ex, NEBNext
        20 mM Tris-HCl
        12 mM (NH4)2SO4
        5 mM MgCl2
        0.16 mM β-NAD
        0.19 mM dNTPs each
        pH 7.4 @ 25°C

        Comment


        • #5
          I think by cleaning up cDNA uniformity and consistency among samples and batches will be increased. Non-cleaned cDNA would have various amounts of left over oligos and salts which may affect library quality.

          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
          7 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          7 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
          66 views
          0 likes
          Last Post seqadmin  
          Working...
          X