DNA Sequencing Reactions using Big Dyes

• Use GFX purified plasmids prepared according to manfacturers instructions. Should be about 0.3 mg plasmid DNA/ml.
  
  
• Add 2.5µl plasmid and 0.5µl of 10µM primer to a 0.2 ml tube or multitube plate well.
  
  
• Add 2.0µl of ABI Big Dye reagent.
  
  
• Ensure all components are at the bottom of the tube by pulse spinning for 15 seconds. Cycle as below:

• Add 14 µl of a mixture of 1ml ethanol, 40µl 3M sodium acetate pH 7.0 and place at -20°C for 10 mins
  
  
• Spin for 15min at full speed in microcentrifuge, or plates for 30mins at max speed in Jouan plate carrier rotor.
  
  
• Remove supernatant by aspiration, or spin plates  upside down for 2min onto tissue paper in Jouan plate carrier.
  
  
• Add 150µl 70% ethanol to each tube and reapeat previous step
  
  
• Allow to air dry in open tubes for 10 mins
  
  
• Resuspend in 4µl of formamide plus ABI loading dye.
  
  
• Mix and load (1.5µl in 48 well, 1µl in 65 well) - do not heat before loading.