Note: Proceed to the next step immediately (if not possible, spin for an extra 2 mins before performing
next step).
• Invert the plate to decant supernatant. Keep the plate inverted and spin up to 185 x g (line the
plate with papertowel), then remove from the centrifuge. The timing starts when the rotor
starts moving.
Note: Supernatant must be removed completely or unincorporated dye terminators will remain in the
samples and interfere with sequencing.
• Add 60ul of freshly made 70% ethanol to each well.
• With the centrifuge set at 4°C, spin at 1,650 x g for 15 min.
• Invert the plate to decant supernatant. Keep the plate inverted and spin up to 185 x g (line the
plate with paper towel), then remove from the centrifuge.
• Cover with aluminum foil, and store at 4°C until submission.
Note: Make sure wells are dry. You may use a speed-vac for 15 mins to dry. Make sure samples are
protected from light while drying.
iii) Purification Method - Ethanol/EDTA Precipitation (1.5ml tubes):
This method is good for removal of excess dye terminators (unincorporated dye labeled terminators).
While this method produces the cleanest signal, it may cause loss of small molecular weight fragments.
To precipitate 20ul sequencing reactions in 1.5ml microfuge tubes:
• Pipette entire contents (20µl) of the extension reaction into a 1.5ml microfuge tube
• Add the following to each well:
5ul of 125mM EDTA, making sure the EDTA reaches the bottom of the wells
60ul of 100% ETOH to each
• Close the tubes and vortex briefly.
• Leave the tubes at room temperature for 15 mins to precipitate the extension products.
Note: Precipitation times shorter than 15 mins will result in loss of very short extension products.
Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye
terminators.
• Place tubes in micro-centrifuge and spin for 20 mins at ≥14,000 x g.
Note: Proceed to the next step immediately (if not possible, spin for an extra 2 mins before performing
next step).
• Carefully aspirate the supernatants with a separate pipette tip for each sample and discard.
Pellets may or may not be visible.