DNA Extraction Protocol - Part 2

Summary

This video provides a step-by-step guide for precipitating DNA from a solution. The process includes adding ethanol to the sample, mixing, and centrifuging to form a DNA pellet. After carefully removing supernatant and washing the pellet with ethanol, the DNA is dried and re-suspended in molecular biology grade water. Key steps involve balancing tubes in the centrifuge, avoiding pellet disruption, and ensuring proper evaporation for complete dryness. The final product is DNA ready for further processing or analysis.

Takeaways

• 😀 Add an equal volume of 100% ethanol to the DNA samples to precipitate the DNA from solution.
• 😀 Mix the solution by inversion 10 times to ensure thorough mixing.
• 😀 Cloudiness in the solution is normal and indicates DNA precipitation.
• 😀 Incubate the samples at room temperature for 5 minutes before centrifuging.
• 😀 Ensure that the microcentrifuge tubes are balanced with hinges facing outward during the spin.
• 😀 After centrifugation, visually inspect for a DNA pellet at the bottom of the tube.
• 😀 If no pellet is visible, you may choose to centrifuge again or proceed as normal.
• 😀 Carefully remove approximately 800 microliters of supernatant using a P1000 pipette without disturbing the pellet.
• 😀 Clean the DNA pellet by adding 250 microliters of 70% ethanol and do not mix to avoid disturbing the pellet.
• 😀 Centrifuge the samples again to remove as much supernatant as possible without disturbing the DNA pellet.
• 😀 Allow the remaining solution to evaporate by leaving the tube open on its side for 20-30 minutes to dry the pellet.
• 😀 Re-suspend the dried DNA pellet by adding 100 microliters of molecular biology grade water and vortexing for a few minutes.

Q & A

Why is ethanol added to the samples during DNA precipitation?

Ethanol is added to precipitate the DNA from the solution, causing the DNA to clump together and form a pellet that can be collected by centrifugation.

What is the purpose of mixing by inversion 10 times?

The purpose of mixing by inversion is to thoroughly combine the ethanol with the DNA solution, ensuring effective precipitation of the DNA.

What should be done if the pellet is not visible after centrifugation?

If the pellet is not visible, you can either centrifuge the sample again or proceed with the next steps, depending on the experiment's requirements.

Why is it important to balance the tubes in the centrifuge?

Balancing the tubes is essential to prevent damage to the centrifuge and ensure proper and even spinning, which is crucial for efficient DNA precipitation.

What is the correct method for removing the supernatant after the first centrifugation?

Using a P1000 pipette, remove approximately 800 microliters of supernatant, being careful not to disturb the DNA pellet. Discard the solution and repeat for each sample.

How should the DNA pellet be cleaned after the first wash?

The DNA pellet is cleaned by adding 250 microliters of 70% ethanol to each sample. It is important not to mix the solution to avoid disturbing the pellet.

What is the purpose of the second centrifugation after washing with ethanol?

The second centrifugation is used to remove residual ethanol and further purify the DNA pellet, ensuring that excess contaminants are eliminated.

How should you remove the supernatant during the second wash step?

Use a P200 pipette for precision to carefully remove and discard the supernatant, ensuring minimal disturbance of the DNA pellet.

Why is it important to allow the tubes to air dry after the final wash?

Air drying allows any remaining ethanol to evaporate, ensuring that the DNA pellet is dry before re-suspension, which is crucial for accurate downstream processing.

How do you re-suspend the DNA pellet after drying?

After the pellet is dry, add 100 microliters of molecular biology-grade water directly to the side of the pellet, cap the tube, and mix by vortexing to dissolve the DNA.