DNA Template

  • Template Preparation, Quality, and Quantity Requirements

    Important: All samples must be labeled with the sample name, date, and user account number. All samples must be accompanied by a sequencing request form indicating the appropriate service package.

    Full-service DNA sequencing begins with the submission of isolated and purified template DNA and primers. Acceptable templates include double and single stranded DNA, PCR products, and genomic DNA.

    Various kits are commercially available to provide investigators with the proper quantity and quality of template for genetic analysis. Investigators should consider the following when choosing an isolation method:

    • Starting Material (cells, tissue, DNA fragments)
    • Throughput (single tube processing or plate format)
    • Expected Yield (mini, midi, maxi preparations)
    • Subsequent Purification

    Quantity
    Guidelines for submission as per Applied Biosystems’ BigDye Terminator Cycle Sequencing protocols:

    Template Quantity/rxn Required concentration
    PCR Product:    

    100-200 bp

    1-3ng 5ng/µL

    200-500 bp

    v3-10ng 5ng/µL

    500-1000 bp

    5-20ng 5ng/µL

    1000-2000 bp

    10-40ng 5ng/µL

    >2000 bp

    20-50ng 5ng/µL
    Single-Stranded 25-50ng 5ng/µL
    Double-Stranded 300ng 100-200ng/µL
    Cosmid, BAC 0.5-1.0 µg 500ng/µL
    Bacterial genomic DNA 2-3µg 1µg/µL
    genomic DNA 50ng 25ng/µL
    *Customer Primers 3-4pmol 1pmol/µL = 1µM

    The concentrations listed are necessary for speedy processing and investigators should plan on providing at least 2X the volume requirements for each sequencing reaction. If your laboratory cannot achieve these concentrations, vacuum dry concentrating is available at the Genomics Core Facility as a custom service. Supplying extra template will ensure that sufficient material is on hand if it is necessary to re-run a reaction. Investigator must provide documentation confirming the concentration of submitted samples.

    * The following common vector primers are kept in stock and available from the Genomics Core Facility: -21M13Forward, M13Reverse, T3, T7promoter, BGH Reverse and Bluescript Primers: KS & SK.

    ** For best results genomic DNA should be subjected to preliminary PCR reactions and purification before cycle sequencing. Please contact us to inquire about this service.

    Quality
    For optimum results, good quality template is essential. For full-service sequencing, the investigator is expected to provide template samples with A260/A280 of 1.8-2.0. We can include a template quality check as a custom service prior to beginning thermal cycling. If quality is insufficient, the investigator will be notified.

    For basic service sequencing, in which samples are provided as purified, lyophilized cycle sequencing products, it is strongly recommended that you perform a quality check at your laboratory, prior to sample submission, to avoid unnecessary delays.