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DNA Sanger Sequencing - Sample Preparation Guidelines
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If you have any questions regarding sample preparation which are not addressed in this document, please contact the Facility at (412) 383-9077 or gpclseq@pitt.edu.

You must be a registered facility user before submitting any samples for processing. If your laboratory is new to our services, please register here.

This page provides information on:
General sample submission
Template preparation
Sample submission for PCR clean-up prior to sequencing
Universal primer addition service
Sample submission for sequencing reaction
Sample submission for capillary run only for sequencing
Sample submission for capillary run for genotyping

General Sample submission guidelines:
  1. Samples may be left at any of our drop off locations, 3343 Forbes Avenue, Room 303, Scaife Hall, room S534A or Hillman Cancer Center Research Pavilion, room 2.15. Please leave samples grouped together in the marked box. At Hillman, please use the individual boxes supplied by GPCL. Please do NOT leave them in bags, racks without lids or attached to the drop off form. Please note that courier service from the Hillman center to the facility at 3343 Forbes Avenue is provided by, and at the convenience, of the Hillman Center and is not in anyway coordinated by the GPCL or its staff.

  2. For samples submitted for capillary run only, please see guidelines below. All other samples can be submitted in single or 8-strip PCR tubes, 0.5 ml tubes or 1.5 ml tubes. If there are more than 48 samples, send a 96-well (conical) plate, DO NOT use cell-culture, round or flat-bottom plates. To reduce evaporation, condensation and cross-well contamination, cover with an adhesive plate cover, NOT parafilm, foil or hard plastic.

  3. LABEL samples with initials and a SINGLE number (i.e. JD1, JD2, ‘¡­.) on the top of the tube. PI name should be written on side. Keep an internal record of sample names; you DO NOT have to send them to us. Label plates on the side with PI name, date of submission and plate number if more than 1 is submitted.

  4. DO NOT use tape anywhere, ever.

  5. Please use the GPCL sequencing and genotyping sample submission form available on our website and leave a completed hard copy in the sample drop off room.

Failure to adhere to these sample submission guidelines may result in sample processing delay or additional charges.

Sequencing Template preparation

Appropriate template preparation is the responsibility of the researcher. For information on the best methods for template preparation, please see Chapter 2, Preparing Templates from ABI BigDye Terminator v3.1 Cycle Sequencing Kit protocol.

Submissions for PCR Clean-up and Sequencing Reaction

  1. It is recommended that PCR products be run on an agarose gel to ensure that the products are clean prior to submission. If there is more than one amplified product, gel-purified product or nested primers will be required.

  2. The sequencing primer should be diluted to 3.2 µM and submitted in a microcentrifuge tube. (Be sure that the tube is labeled with the PI name, primer name and concentration.) If universal primers are to be used, please see below.

  3. On the sample drop off form, select PCR Clean up and Sequencing reaction as level of service and indicate in the table provided which primers should be used with each of your samples.

 Submissions for Universal Primer Addition and Sequencing Reaction

  1. We can add our universal primers: M13F, M13R, SP6, T3 or T7. On the sample drop off form, in addition to other services requested, select Addition of GPCL provided universal primers and indicate clearly in the space provided which samples should be treated with each primer.

Submissions for Sequencing Reaction

  1. For each reaction the following should be mixed prior to submission:

    X µL of DNA template (determined by the concentration of the template and the table below)
    3.2 pmol of primer,
    Y µL of H20 (to bring total volume to 15 µL)
    15 µL total volume

    The table below shows the recommended template quantity based on template size:

    Template PCR Product of:Template Quantity
    100-200 bp2-6 ng
    200-500 bp2-20 ng
    500-1000 bp10-40 ng
    1000-2000 bp20-80 ng
    >2000 bp80-200 ng
    Plasmid DNA1 µg
    BAC6 µg

 Submissions for Capillary Run - Sequencing

  1. Sequencing reactions must be carried out in 96-well plates from ABI (catalog # N8010560) or the Biological Sciences Stockroom (catalog # PL223).

  2. All sequencing reactions must be done using ABI BigDye Terminator v3.1 chemistry and following ABI's protocols.

  3. The sequencing reactions must be cleaned up, i.e. unincorporated dye terminators must be removed, prior to submission. The most common methods for clean-up are ethanol precipitation, magnetic beads, e.g. Agencourt CleanSEQ or spin columns. The method you choose will depend on your preference and available equipment. For optimum instrument performance, indicate the clean up method used in the space provided on the drop off form.

  4. Plates must be brought to the Facility after the final drying step of the clean-up. Cover plates with adhesive plate sealers (NOT parafilm, foil or hard plastic).

  5. Electronic sample sheets must be prepared using the template provided (open in Excel to complete, save as tab delimited text) and sent to gpclseq@pitt.edu.

Submissions for Capillary Run Genotyping

  1. For best results, please contact GPCL with specifics of your particular project before submitting samples for genotyping analysis.

  2. Submit 5-10 µl PCR product in 96 well plates.

  3. Complete and email electronic sample sheet.

  4. Complete sample drop off form. Please indicate what type of standard is used and whether you have added it or wish GPCL to do so. Also indicate whether multiple PCR plates should be pooled into a single capillary run.