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- 4°C Microcentrifuge (or normal microcentrifuge in cold room).
Procedure
- Transfer DNA to a 1.7 ml Eppendorf tube. If your sample volume is < 200 µl, it can be helpful to add dH2O to reach 200 µl.
- Add a volume of Sodium Acetate equal to one tenth the sample volume. This provides the high ion concentration and right pH for the DNA to precipitate.
- Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to –20°C.
- Add 2 µl linear acrylamide (10 µg) to the sample, mix, and let stand in –20°C freezer for at least 30 minutes. In some cases longer in the freezer or overnight may improve recovery.
- Centrifuge 14,000×g for 15 minutes at 4°C.
- Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
- Add 200µl of cold 70% ethanol. Pipet up and down until pellet dislodges from the side of the tube. Centrifuge for 10 minutes at 4°C.
- Remove supernatant with a 200µl pipet. Evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37°C heat block.
- Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].
* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 150µl 100% EtOH, and 2 µl acrylamide.
Variation: When precipitating single-stranded nucleic acid samples it may improve recovery to resuspend in 0.001% SDS to prevent sticking to the wall of the tube. |