Announcement

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

  • Bisulphite Sequencing Proof-reading or NOT proof-reading?

    I'm in the process of applying bisulphite PCR amplicon sequencing to the Illumina platform and wondered what peoples thoughts were on whether to use a proof-reading enzyme in the PCR that can read through Uracil's on a bisulphite converted template (I have only come across these two that apparently do):

    pfu TURBO Cx Hotstart (Stratagene)
    Expand High Fidelity PLUS (Roche)

    OR

    Do I continue with the standard hotstart Taq we use in the lab for most bisulphite PCR applications which we know to work very well and gives good amplification products (also I have achieved a good optimisation of all my amplicons with this standard Taq).

    Some people say that - With the read depth provided by GAII and above, the level of fidelity of standard Taq should not have a major impact on your results. While there is a tenancy to use a proof-reading enzyme in all commercial sequence capture protocols.

    Please help - any comments would be grateful

  • #2
    I recently went through this protocol and used pfu turbo polymerase. I initially tried PCRing with the taq provided in the truseq kit which I believe is phusion polymerase but I could be wrong. That didn't work and I assumed it was because of the U's. But I guess if you have gotten the normal hotstart Taq to work for previous bisulfite products then that wouldn't be an issue for you.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Advanced Tools Transforming the Field of Cytogenomics
      by seqadmin


      At the intersection of cytogenetics and genomics lies the exciting field of cytogenomics. It focuses on studying chromosomes at a molecular scale, involving techniques that analyze either the whole genome or particular DNA sequences to examine variations in structure and behavior at the chromosomal or subchromosomal level. By integrating cytogenetic techniques with genomic analysis, researchers can effectively investigate chromosomal abnormalities related to diseases, particularly...
      09-26-2023, 06:26 AM
    • seqadmin
      How RNA-Seq is Transforming Cancer Studies
      by seqadmin



      Cancer research has been transformed through numerous molecular techniques, with RNA sequencing (RNA-seq) playing a crucial role in understanding the complexity of the disease. Maša Ivin, Ph.D., Scientific Writer at Lexogen, and Yvonne Goepel Ph.D., Product Manager at Lexogen, remarked that “The high-throughput nature of RNA-seq allows for rapid profiling and deep exploration of the transcriptome.” They emphasized its indispensable role in cancer research, aiding in biomarker...
      09-07-2023, 11:15 PM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 09-29-2023, 09:38 AM
    0 responses
    10 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-27-2023, 06:57 AM
    0 responses
    12 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-26-2023, 07:53 AM
    0 responses
    30 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-25-2023, 07:42 AM
    0 responses
    18 views
    0 likes
    Last Post seqadmin  
    Working...
    X