ABI BigDye Terminator sequencing

Instructions for cycle sequencing of PCR products and plasmids using the ABI BigDye Terminator cycle sequencing kit. P/N 4303149
Template concentration Cycling Conditions
PCR Products: 1.  95 C   2 m
2 ng/ul/100bp of product 2.  96 C   5 s
Plasmids: 3.  50 C   5 s
200 ng/ul for a 3-5 kb plasmid 4.  60 C   4 m
Primer concentration:

35 cycles
3 uM
Reaction Setup Formamide/Crystal violet loading dye (1000 X)
4 ul ABI BDT mix 0.5 -1 g crystal violet
2 ul  primer (concentration above) 50 ml nanopure water
4 ul template (concentration above) dilute 1:1000 with deionized formamide to make 1X dye
10 ul total volume
Reaction cleanup

Excess terminators and salts  need to be removed from the reaction before running on a gel in one of two ways.
Isopropanol Precipitation Edge Biosystems filtration cartridges # 42453
  • Add 40 ul (4 volumes) of 75% isopropanol (24-26 C) to the sequencing reaction
  • cover and mix by inverting
  • leave at 24-26 C for 15 minutes
  • spin in a balanced microplate carrier of the Eppendorf 5804 centrifuge at maximum speed for 40 min. keep the temp. at 24 C.
  • immediately after centrifugation, uncover plate and gently invert onto a paper towel.
  • place in centrifuge upside-down on a paper towel and spin at 500 x g for 1 minute.
  • Centrifuge cartridge as supplied for 2 minutes at 750 x g.
  • Transfer Cartridge to a labeled clean microfuge tube.
  • Add sequencing reaction to the center of the matrix.
  • Close the cap and centrifuge for 2 minutes at 750 x g.
  • Discard cartridge.
  • Dry contents of tube in speed vac. at low setting for 15-20 minutes until liquid is gone.
  • reconstitute sample in 20 ul of 1X formamide/crystal violet loading dye.
  • denature samples for 2 minutes at 95 C and place on ice prior to loading.