DNA Extraction Protocol - Part 1

Summary

This instructional video guides students through a DNA extraction protocol, ensuring proper lab flow to avoid contamination. It covers the steps of transferring saliva samples, incubating them with a purifying solution, vortexing, and centrifuging the samples to isolate DNA. The procedure emphasizes careful handling, proper tube labeling, and maintaining sample integrity. After centrifugation, the DNA-containing supernatant is carefully transferred to new tubes for further analysis. This method ensures high-quality, contaminant-free DNA preparation for subsequent genotyping.

Takeaways

• 😀 Ensure proper workspace flow to prevent contamination, with tips and pipettors on the left, samples in the center, and discard bucket on the right.
• 😀 Avoid crossing your hands over samples during the protocol to maintain cleanliness and reduce the risk of contamination.
• 😀 Begin the process after students have spit into DNAgenotek collection kits, capped tubes to release the buffer, and mixed by inversion.
• 😀 Do not label sample tubes until after mixing to ensure student anonymity during genotyping.
• 😀 When using a P1000 pipettor, draw the solution slowly to avoid bubbles or contamination, and always close the sample tube after transferring liquid.
• 😀 Preheat the heat block to 50°C and turn it on before placing the samples inside, and incubate for at least 90 minutes or overnight.
• 😀 Add DNAgenotek's proprietary purifying solution, PrepIT, to the incubated samples, and mix with a vortex to ensure proper mixing.
• 😀 After vortexing, place samples on ice for 10 minutes before proceeding with centrifugation to remove impurities.
• 😀 Balance the samples evenly in the microcentrifuge, ensuring all tubes have equal volumes before closing the lid and starting the centrifugation process.
• 😀 After centrifugation, transfer 400 microliters of supernatant (containing DNA) to a new tube, being careful not to disturb the pellet formed at the bottom of the tube.

Q & A

Why is it important to ensure the workspace has a proper flow before starting the protocol?

A proper workspace flow prevents contamination of samples or reagents, ensuring accurate and reliable results in the experiment.

Why is the sample tube not labeled until after the student samples are mixed?

The sample tubes are not labeled initially to maintain student anonymity during the genotyping process, preventing any bias in handling the samples.

What should you be cautious of when pipetting large volumes?

When pipetting large volumes, you should draw up the solution slowly to avoid creating bubbles or contaminating the micro-pipettor.

What is the purpose of placing the samples in the heat block set to 50 degrees?

The heat block is used to incubate the samples for at least 90 minutes, which helps in preparing the samples for further processing by facilitating chemical reactions or enzymatic activities.

What happens when you mix the sample with the purifying solution and why?

When the sample is mixed with the purifying solution, it may become cloudy. This is a normal reaction, and the solution is then mixed with a vortex to ensure that the components are well-incorporated.

Why is it important to embed all samples in ice for 10 minutes?

Embedding the samples in ice for 10 minutes helps in removing impurities by freezing certain components, preparing the sample for centrifugation.

What is the correct way to balance tubes in the centrifuge?

The tubes should be balanced across the rotor by ensuring that they contain equal volumes. This prevents unbalanced spinning and reduces the risk of damaging the centrifuge.

What should you do if the centrifuge vibrates violently during the spin?

If the centrifuge vibrates violently, it indicates that the tubes are unbalanced or the internal lid has come loose. In this case, you should press the Stop button immediately to avoid damage.

How do you handle the sample after centrifugation to avoid disturbing the pellet?

After centrifugation, you should remove the internal lid and carefully remove the samples, ensuring that the pellet at the bottom of the tube is not disturbed to preserve the integrity of the sample.

What should you do with the supernatant after centrifugation?

The supernatant, which contains the DNA, should be transferred to a new tube, avoiding the pellet at the bottom of the tube to ensure that only the DNA-rich liquid is collected.