Sanger Sequencing Reaction

Procedure

• In a 0.2 ml PCR tube add
  • template 5.0 ul (200 ng/ul)
  • VF primer 1.0 ul
  • dilute buffer 2.5 ul (reads BD dilute buffer on tape of cap) in PCR/Sequencing box
  • BD sequence mix 1.5 ul (reads BD)
• Mix well.
• Select program 'seq-dye' on PTC 200 thermal cycler
• Run program. It will take about 2 hrs.
• Remove tube from PCR machine and transfer 10 ul rxn mix to 1.5 ml Eppendorf tube. Add
  • 1.5 ul Blue NaOAc/EDTA
  • 40 ul 95% ethanol
• Let sit on ice 15 min
• Centrifuge 10 min max speed
• Should see a small blue dot at bottom of tube
• Discard supernatant
• Wash pellet with 500 ul 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 min
• Place in -20 C freezer to be delivered to MBSU 4th floor microbiology M534. Rxns delivered before 2pm will normally be returned the next day.