NOTE: When you sequence, you sequence in both forward and reverse directions. But this is done with two different reactions. Each reaction should only have forward or reverse primer in it, not both. For one plate, two plates must be created for sequencing, one with the forward primer and one with the reverse primer.

  1. Make a bit more sequencing mix than you need for your reactions, so for a plate of 96 samples, make enough sequencing mix for 100 samples.
  2. For very bright bands on the E-gel, use only 1ul of nondiluted PCR product. For faint bands use 1.5ul PCR product.
  3. Add the primer and PCR product to a non-skirted plate (or to an eight-well strip if you are sequencing only a few samples).
  4. Make a master mix of BigDye, 5x SeqBuffer, and ddH2O in a 1:1:10 ratio. Vortex the mix.
  5. Add 12ul of the master mix to each well.
  6. Label the plate and then seal it with self-adhesive foil labeled with your name, the primer, and the date(or cap strips if using eight-well strips).
  7. Run in the thermocycler using the program Seq31.
  8. Fill out the submission form and e-mail it to Jeff at The forms can be found at
  9. Get Bob to sign the form and write a grant number before you give the form and sequencing plate to Jeff in the Genomics Facility.
  10. Jeff will e-mail you the sequences if there are only a few. If there are many, then Jeff will e-mail you telling you when you can go to the Genomics Facility with a USB key to pick up the sequences.

For sequencing in the Genomics Facility each reaction requires:

  • Big Dye 1 ul
  • 5X SeqBuffer 1 ul
  • ddH2O 10 ul
  • Primer (10 pmol) 1 ul
  • About 1-1.5ul PCR template

The next step is Sequence Editing with SeqScape or Sequence Editing with Sequencher.

Return to Barcoding in the Hanner Lab Wiki.

Updated May 2, 2009