MyGPCL Login  |  Register    
   Register    Directory     Protocols    Forms     Pricing    Open Positions       
DNA Sanger Sequencing - Sample Preparation Guidelines
Services | DNA Sequencing Services | Sample Submission

If you have any questions regarding sample preparation which are not addressed in this document, please contact the Facility at 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, Hillman Cancer Center Research Pavilion, room 2.15 or Children’s Hospital loading dock. Please leave samples grouped together in the marked Sequencing box. At Children’s, samples must be placed in the insulated container for Dynamex. Samples loose in the fridge will not be collected. At Hillman, please use the individual boxes supplied by GPCL. Please do NOT leave samples 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 any way 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 1.5 ml tubes containing 15 µl of the template-primer mix as described below. If there are more than 24 samples, send 6 µl total volume in a 200-300 µl 96-well (conical) semi-skirted PCR compatible plate. DO NOT use cell-culture, round or flat-bottom plates. We recommend ABI (catalog # N8010560) or the Biological Sciences Stockroom (catalog # PL223).

  3. To reduce evaporation, condensation and cross-well contamination, cover plates with an adhesive plate cover, NOT parafilm, foil or hard plastic.

  4. LABEL sample tubes with initials and a SINGLE number (e.g. JD1, JD2, ...JD12, JD13....) on the top of the tube. PI name should be written on side. Keep an internal record of sample names; 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 one is submitted.

  5. Partial plates must be filled contiguously in column order, i.e. A1 through H1 then A2 through H2. Empty wells in the middle of a group will not be skipped and you will be charged for a reaction. Plates filled in row order will be returned for rearrangement prior to processing.

  6. DO NOT use tape anywhere, ever.

  7. 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.

  8. When submitting samples for sequencing reaction in plates, an Electronic Instrument Sample Sheet must be prepared for each plate using the SEQ template available on our forms page and sent to gpclseq@pitt.edu:

    1. Open template in Excel to complete
    2. Enter "plate name" in cells A2 and B2
      1. Name must be unique and concise
      2. Name must match Sample Sheet filename
      3. Match filename to the 96-well plate name for ease in identification
      4. DO NOT USE SPACES
      5. Use only alphanumeric characters, underscore or dash
    3. Enter Sample IDs in Column B
      1. Use only alphanumeric characters, underscore or dash
      2. USE NO SPACES (check for blank spaces after Sample ID)
      3. Do not leave any empty cells in the spreadsheet. Enter ´empty¡ to designate empty wells. d.Save as tab delimited text file (.txt)
    4. Email completed Instrument Sample Sheet(s) to gpclseq@pitt.edu

    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 single bands 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 provide addition of the following universal primers free of charge: 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. Failure to mark this option may result in sample processing delay.

  2. If submitting less than 24 samples, please submit 15 µl of template in 1.5 ml tubes to allow for accurate transfer and timely repeat in the event of technical failure. If you are requesting that samples be run with 2 different primers, please provide 2 separate tubes of template.

  3. If submitting more than 24 samples, please submit 5 µl of template in a 200-300 µl 96 well semi-skirted PCR compatible (conical) plate for addition of universal primers prior to sequencing reaction. If you are requesting that samples be run with 2 different primers, please provide 2 separate wells of template.

  4. See table below for recommended template mass to be submitted based on template size.

Submissions for Sequencing Reaction

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

    1. If submitting less than 24 samples in 1.5 ml tubes:

      X µL of DNA template (determined by the concentration of the template and the table below)
      8.0 picomoles of primer (absolute mass not concentration)
      Y µL of H20 (to bring total volume to 15 µL)
      15 µL total volume

    2. If submitting more than 24 samples in a semi-skirted 200-300 µl 96-well PCR plate:

      X µL of DNA template (determined by the concentration of the template and the table below)
      3.2 picomoles of primer Y µL of H20 (to bring total volume to 6 µL)
      6 µL total volume

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

      Template PCR Product of:Template Quantity in 15µl tube submissionsTemplate Quantity in 6µl tube submissions
      100-200 bp2-6 ng1-3 ng
      200-500 bp6-20 ng3-10 ng
      500-1000 bp10-40 ng5-20 ng
      1000-2000 bp20-80 ng10-40 ng
      >2000 bp80-200 ng40-100 ng
      Plasmid DNA1 µg500 ng
      BAC6 µg3 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. An Electronic Instrument Sample Sheet must be prepared for each plate using the SEQ template available on our forms page. Open the provided template in Excel, fill out as specified below, save as a tab delimited text file and send to gpclseq@pitt.edu.
    1. Open template in Excel to complete
    2. Enter "plate name" in cells A2 and B2
      1. Name must be unique and concise
      2. Name must match Sample Sheet filename
      3. Match filename to the 96-well plate name for ease in identification
      4. DO NOT USE SPACES
      5. Use only alphanumeric characters, underscore or dash
    3. Enter Sample IDs in Column B
      1. Use only alphanumeric characters, underscore or dash
      2. USE NO SPACES (check for blank spaces after Sample ID)
      3. Do not leave any empty cells in the spreadsheet. Enter "empty" to designate empty wells.
    4. Save as tab delimited text file (.txt)
    5. Email completed Instrument Sample Sheet(s) 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 10 µl PCR product in 96-well semi skirted plate.

  3. To reduce evaporation, condensation and cross-well contamination, cover plates with an adhesive plate cover, NOT parafilm, foil or hard plastic.

  4. Label plates on the side with PI name, date of submission and plate number if more than one is submitted.

  5. An Electronic Instrument Sample Sheet must be prepared for each plate using the GENOsample sheet available on our forms page. Open the provided template in Excel, fill out as specified below, save as a tab delimited text file and send to gpclseq@pitt.edu:

    1. Open template in Excel to complete
    2. Enter "plate name" in cells A2 and B2
      1. Name must be unique and concise
      2. Name must match Sample Sheet filename
      3. Match filename to the 96-well plate name for ease in identification
      4. DO NOT USE SPACES
      5. Use only alphanumeric characters, underscore or dash
    3. Enter Sample IDs in Column B
      1. Use only alphanumeric characters, underscore or dash
      2. USE NO SPACES (check for blank spaces after Sample ID)
      3. Do not leave any empty cells in the spreadsheet. Enter "empty" to designate empty wells.
    4. Save as tab delimited text file (.txt)
    5. Email completed Instrument Sample Sheet(s) to gpclseq@pitt.edu
  6. Complete sample drop off form and submit a hard copy along with your samples.
    1. Please indicate what type of standard is used (LIZ or ROX) and whether you have added it or wish GPCL to do so.
    2. Indicate whether multiple PCR plates should be pooled into a single capillary run.
    3. Map Marker standard must be present in each well. You may provide an unopened bottle and request GPCL add it to your samples, or GPCL can provide LIZ GeneScan 500.