Ethanol Precipitation Protocol

Overview

The method described in this protocol for precipitating sequenced product is derived from the ABI Ethanol Precpitation Protocol. There are four basic steps:

  1. Combine the four sequenced products made from A, C, G, and T rxn mix into a nunc plate. The nunc plate is


  2. used because the circumference of its well allows the 384-split-well plate to fit nicely into it.


  3. Add the 95% ethanol and then mix well.


  4. Spin down the precipitated product and discard the supernatant.


  5. Allow the precipitated product to dry, then add deionized water to reconstitute it. Deionized water is used because it is less damaging to the 3700's capillaries than Formamide.

Equipment

  • Multimek 96 Automated 96-channel Pipettor (Beckman Instruments)
  • Jouan Centrifuge (CR422)
  • Rigid Thermo-fast plates, 96-well (Island Scientific) (referred to as 96-well plates)
  • 96-well polypropylene
  • plate V BTM (Nalge Nunc International) (referred to as nunc plate)
  • Freezer

Reagents

  • 95% Ethanol
  • Sequenced PCR Product (Setup in a 384 split-well plate)
  • Thermowell sealer (aluminum) for 96-well plates

Protocol

  1. Centrifuge the sequenced 384 split-well plate at 1000rpm (or 190g) for 20 sec (Program 1).


  2. Place the nunc plate on top of the 384 split-well plate, then turn them over.


  3. Centrifuge the two combined plates at 3500rpm for 45 sec (Program 4).


  4. Label a Rigid Thermo-fast plate into which the ethanol and the sequenced product will be transferred. Since the


  5. 384 split-well plate was flipped onto the nunc plate, label the 96-well plate accordingly (i.e. the left hand product on the sequencing plate will be labelled on the right side of the unskirted 96-well plate and the right hand product will be labelled on the left hand side of the unskirted 96-well plate).


  6. Remove the 384-split-well plate from the nunc plate


  7. Add 33 ul of 95% ethanol to the nunc plate by hand. Use the program "Ethanol Precipitation V.3" on the Multimek Robot to slowly mix the ethanol with the sequenced products 20 times and transfer the mixture from the nunc plate to the labelled 96-well Rigid Thermo-fast plate.


  8. Use the program Ethanol Precipitation V.3 on the Multimek Robot to add 54 ul of 95% Ethanol to the nunc plate. The Multimek Robot will slowly mix the Ethanol with the sequenced products 30 times and will transferthe mixture from the nunc plate to the 96-well plate.


  9. Place a Thermowell sealer on top of the 96-well plate and allow the plate to sit in the freezer for 15-20 min.


  10. 9.Remove the Thermowell sealer. Using the centrifuge, spin the 96-well plate at 3500rpmor (2350g) for 45 min.


  11. After 45 min, turn the 96-well plate over and place a paper towel on the bottom of the carrier. Then spin the plate at 500rpm or (47g) for 12 sec.


  12. Allow the 96-well plate to air dry for 30 min. At this point, the sample can be sealed with a Thermowell sealer and stored in the freezer. Add 10 l of deionized water to each of the 96 wells before transferring to a 384-well plate for loading (see "Preparation of 384-well plates for sequencer loading).


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