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L A B O R A T O R Y
Part V: DNA Purification and Automated Sequencing

This procedure isolates the mt amplicon from unincorporated NTPs.
Reagents Equipment & Supplies Shared Items
3M sodium acetate (pH 5.2), 1µl
95% ethanol, 30 µl
Cycle sequencing product, 10µl
70% ethanol, 100 µl
Ice
H2O
test tube, polypropylene
1-20 µl micropipet and tips
 Vortex
95° C waterbath or heat block

Pre-lab Prep

Prepare loading dye of 5 parts deionized formamide to 1 part 50 mM EDTA w/blue dextran.

Procedure

  1. Remove sealing foil from plate.
  2. Add to each well containing sample: 1 µl of 3 M sodium acetate, pH 5.2 and 30 µl of 95% ethanol.
  3. Cover plate using sealing foil.
  4. Vortex plate and place on ice for 30 minutes.
  5. Centrifuge for 30 minutes. (use a second 96-well plate as a balance)
  6. Remove sealing foil and invert plate onto paper towel. Gently tap until most of the ethanol solution has been removed.
  7. Rinse pellet by adding 50 µl of 70% ethanol into each well. Cover plate with sealing foil and centrifuge for 15 minutes.
  8. Remove sealing foil and invert plate onto paper towel. Gently tap until most of the ethanol has been removed.
  9. Rinse pellet again by repeating steps 7 and 8.
  10. If possible, let the plate air dry overnight.
  11. After the plate is thoroughly dry, resuspend all wells containing sample with 20 µl of dH2O.
  12. Load 96-well plate onto automated sequencer (ABI 3700 DNA Analyzer).
2000, DNA Learning Center, Cold Spring Harbor Laboratory
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