Overview
This lecture covers the step-by-step protocol for precipitating, washing, and re-suspending DNA from solution using ethanol and centrifugation.
DNA Precipitation
- Add an equal volume of 100% ethanol to each DNA sample to precipitate the DNA.
- Mix the solution by inverting the tube 10 times; cloudiness may appear.
- Incubate samples at room temperature for 5 minutes.
Centrifugation and Pellet Identification
- Place tubes in a microcentrifuge with hinges facing outward, ensuring tubes are balanced.
- Centrifuge the samples for 2 minutes.
- Carefully remove tubes and check for a DNA pellet at the bottom near the hinge; a pellet may not always be visible.
Supernatant Removal and Ethanol Wash
- Remove and discard ~800 µL of supernatant using a P1000 pipette, avoiding the pellet.
- If the pellet is missing, note this occurrence.
- Add 250 µL of 70% ethanol to each tube to wash the pellet; do not mix.
- Centrifuge again for 2 minutes.
Final Removal of Supernatant and Drying
- Carefully remove as much supernatant as possible with a P200 pipette, using multiple draws if needed.
- Knock the tube onto a Kimwipe to remove any large drops.
- Leave the tube open and on its side to air dry for 20-30 minutes, checking for remaining moisture.
DNA Re-Suspension
- Add 100 µL of molecular biology grade water directly onto the dry pellet.
- Cap the tube and vortex for several minutes to re-suspend the DNA.
- Visually confirm the pellet has dissolved or re-suspended; if not, continue vortexing or allow to sit at room temperature.
Key Terms & Definitions
- Ethanol Precipitation — A method to isolate DNA by adding ethanol, causing DNA to become insoluble and form a pellet.
- Supernatant — The liquid above the pellet after centrifugation.
- Pellet — The small mass of DNA collected at the bottom of a centrifuged tube.
- Centrifuge — A machine used to spin samples rapidly to separate components by density.
Action Items / Next Steps
- Ensure proper documentation of any missing pellets during the process.
- Proceed to subsequent DNA analysis or storage steps after re-suspension.