Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

Big Dye Terminator Sequencing

Reagents:

  • Big Dye Terminator kit from Perkin Elmer

  • Gene specific primers (need 3.2 pmol/reaction)

  • Sample DNA:

    • 200-300 ng for dsDNA
    • 75-100 ng for ssDNA
    • 90-150 ng for PCR products

  • ddH2O

  • Isopropanol

  • Mineral Oil

Protocol:

  1. Quantify DNA or PCR product using the Spectrophotometer. The protocol applies to a full reaction. In case of a half reaction, divide all volumes in half but DO NOT decrease DNA or primer amounts.

  2. Mix sample DNA and specific primers to 12 µl total volume.

  3. Add 8 µl Terminator Ready Reaction mix.

  4. Mix well and spin briefly (do not vortex).

  5. If thermal cycler does not have a heated lid, overlay the reaction mix with 40 µl mineral oil.

  6. Put the tubes in thermal cycler and set volume to 20 µl.

  7. Program cycle below and repeat for 25 cycles:

    1. In a GeneAmp 9600/9700 or 2400

      1. 96°C for 10 seconds

      2. 50°C for 15 seconds

      3. 60°C for 4 minutes

      4. 4°C soak until needed

    2. In a Thermal Cycler (TC1) or in a DNA Thermal Cycler 480

      1. 96°C for 30 seconds

      2. 50°C for 15 seconds

      3. 60°C for 4 minutes

      4. 4°C soak until needed

  8. Spin for 1 minute to remove condensation.

  9. Add 20 µl ddH2O and 60 µl 100% Isopropanol, for 100 µl final volume. (Half reactions: add 30µl ddH2O instead of 20 µl.)

  10. Precipitate at room temperature for 20-30 minutes.

    1. Strip well or 96-well plates:

      1. Spin for 30 minutes at 3000x g

      2. Remove from centrifuge and turn upside down on paper towel

      3. Spin for 1 minute at 700x g to remove residual Isopropanol

      4. Remaining Isopropanol: dry in 37°C oven for 2 minutes (no longer).

    2. Individual tubes:

      1. Spin for 20 minutes at 14000 rpm

      2. Remove the Isopropanol after spinning

      3. Add 250 µl 75% Isopropanol, vortex briefly

      4. Spin for 5 minutes at 14000 rpm

      5. Remove Isopropanol.

      6. Dry in heating block.

  11. Fill out sequencing data form.

  12. Put the samples in the -20°C freezer by the DNA Sequencing facility.