Prepare ethanol/sodium acetate mix.
1 x ethanol/sodium
acetate mix
1.5 ul 2.7M
sodium acetate, pH 5.2
34.5 ul 100% ethanol
100 x ethanol/sodium acetate mix
150 ul 2.7M
sodium acetate, pH 5.2
3450 ul 100% ethanol
Add 5 ul of 25 mM EDTA to the reaction mix. Make sure that
EDTA reach the bottom of the tube.
Add 36 ul of ethanol/sodium acetate mix. Seal the plate and
mix by inverting 4 – 5 times. Incubate at RT for 15 min.
Spin at 2000-3000 x g for 30 min. Proceed to the next step
immediately. If this is not possible, then spin the plate for 2 minutes
immediately before performing the next step.
Invert the plate on paper towel and spin up to 185 × g, then
remove from the centrifuge.
Add 60 µL of 70% ethanol to each well. Seal the plate and
mix by inverting 4 – 5 times.
With the centrifuge set to 4 °C, spin at 1650 × g for 15
min.
Invert the plate and spin up to 185 × g for 1 min, then
remove from the centrifuge.
Note: Start timing when the rotor starts moving.
Resuspend the samples in 12 ul of deionized formamide. Load
into ABI fragment analyzer.