Sequencing Reaction Cleanup

  Sequencing Reaction Cleanup Ethanol/EDTA Precipitation (10 µl reaction volume)

  • Remove 96-well ABI plate from thermocycler and pulse spin @1000 rpm
  • Add 2.5 µl of 125 mM EDTA to each well
  • Add 30ul of 100% ethanol to each well
  • Seal tightly with plate seal; mix by inverting 4X
  • Incubate 15 min at room temp
  • Centrifuge 4000 rpm (3000 rcf) for 15min at 4°
  • Immediately* pour off EtOH supernatant with a quick inversion and gentle shake
  • Add 30 µl of 70% EtOH to each well and centrifuge at 3000 rpm (1690 rcf), for 15 min at 4°
  • Immediately* Pour off supernatant and invert on tri-folded paper towel
  • Place in centrifuge (inverted on paper towel) and pulse spin up to 200 rpm to remove excess EtOH (stop the spin when speed is ~ 150 rpm, as the machine will continue to accelerate briefly after the pulse is stopped - DO NOT ALLOW SPEED TO GO HIGHER THAN 500 rpm)
  • Dry sample plates (right side up) under hood in the dark for 1 h.

*Otherwise spin an additional 2 min

If you are unable to deliver samples to AGTC or the Drop Box immediately, then seal and store as a dry pellet at -20°