Big Dye Terminator Precipitation Protocol

Overview

This protocol for precipitating sequenced product is derived from the ABI ethanol precipitation protocols. This method is similar to the Big Dye Primer Precipitation Protocol, with a couple of differences:

  1. The current protocol calls for a final ethanol concentration of 60% (after water is added to the reaction mix), compared with 95% in the Big Dye Primer protocol.
  2. The precipitant needs to be desalted by adding 70% ethanol to the precipitated product. This is necessary because the 3700 DNA sequencing analyzer is very sensitive to excess salt, which might interfere with the individual base peak.

Equipment

Reagents

Protocol

  1. Spin down the 96-well plate
  2. Add 8 (l of deionized water to the sequenced product
  3. Add 32 (l of 95% ethanol to the sequenced product (final concentration of ethanol sould be 60%)
  4. Using the centrifuge, spin the 96 well-plate at 1000rpm or (190g) for 20 sec
  5. Use the Multimek to mix the ethanol and the sequenced product wellusing the program BDT precipitation
  6. Cover the plate with a sheet of thermowell sealer (aluminum) and put the plate in the freezer for 15 mins
  7. After 15 min, spin the plate at 3500rpm or (2350g) for 45 min
  8. After 45 min, turn the plate over and place a paper towel on the bottom of the carrier; then spin the plate at 500rpm or (47g) for 12 sec
  9. Gently add 75 (l of 70% ethanol, then place a paper towel on the bottom of the carrier and spin the plate at 500rpm or (47g) for 12 sec
  10. Allow the plate to air dry for 30 min at room temperature
  11. Add 10 ul of deionized water to each of the 96 wells for loading