Fluorescent Sequencing Reaction


  • In a 0.2ml PCR tube, add:
Template 5.0ul (200 ng/ul)
VF primer 1.0ul
Dilute buffer 2.5ul
BD sequence mix 1.5ul
  • Mix well.
  • Select program 'seq-dye' on PTC 200 thermal cycler.
  • Run program. It will take about 2 hours.
  • Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
Blue NaOAc/EDTA 1.5ul
95% ethanol 40ul
  • Let sit on ice 15 minutes.
  • Centrifuge 10 minutes at max speed.
    • Should see a small blue dot at bottom of tube.
  • Discard supernatant.
  • Wash pellet with 500ul of 70% ethanol.
  • Discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip.
    • Do not disturb the pellet.
  • Air dry for 10 minutes.
  • Place in -20oC freezer to be delivered to MBSU 4th floor microbiology M534. Reactions delivered before 2pm will normally be returned the next day.