Nucleic Acid Isolation from Plant Soft Tissues

Summary

This video provides a step-by-step guide for isolating plant nucleic acids from soft tissues. The process includes cooling grinding equipment with liquid nitrogen, cutting and freezing plant tissue, and grinding it into a fine powder. The sample is mixed with lysis buffer, incubated, and centrifuged to collect the supernatant. After adding ethanol and mixing, the sample is applied to a spin column for purification. The protocol also offers an alternative method using magnetic bead purification. This comprehensive procedure ensures efficient and accurate nucleic acid extraction from plant material.

Takeaways

• 😀 Ensure all grinding equipment and tubes are cooled with liquid nitrogen before starting.
• 😀 Cut soft tissues from the selected plant material and place them into liquid nitrogen.
• 😀 Have a sufficient amount of liquid nitrogen for grinding the sample effectively.
• 😀 Grind the plant tissue into a fine powder using a pestle.
• 😀 Collect the powdered plant sample and store it either in liquid nitrogen or at -70°C.
• 😀 Prepare the lysis buffer and weigh the lysis tube with the buffer.
• 😀 Add up to 50 milligrams of the powdered plant sample to the lysis buffer and mix immediately by vortexing.
• 😀 Lyse the sample by incubating it on a thermal mixer.
• 😀 Centrifuge the sample at maximum speed and carefully collect the supernatant without aspirating the cell debris.
• 😀 Transfer the supernatant to a new tube, add 95% ethanol, and mix thoroughly by pipetting.
• 😀 Apply the sample onto a spin column or use a magnetic bead purification protocol as an alternative.

Q & A

Why is it important to cool the grinding equipment and tubes with liquid nitrogen before starting the plant nucleic acid isolation process?

Cooling the grinding equipment and tubes with liquid nitrogen helps to prevent degradation of nucleic acids during the grinding process and ensures the plant tissue stays frozen, making it easier to grind into a fine powder.

What is the purpose of grinding the plant tissue into a fine powder?

Grinding the plant tissue into a fine powder increases the surface area, allowing for more efficient extraction of nucleic acids and better interaction with the lysis buffer.

Why is it necessary to store the powdered sample in liquid nitrogen or at -70°C?

Storing the powdered sample in liquid nitrogen or at -70°C helps to preserve the integrity of the nucleic acids and prevents any degradation before the lysis and extraction steps.

What is the role of the lysis buffer in the nucleic acid isolation process?

The lysis buffer is used to break open the plant cells, releasing their nucleic acids and other cellular contents into solution for further purification.

Why should you mix the sample immediately after adding it to the lysis buffer?

Mixing the sample immediately after adding it to the lysis buffer ensures thorough interaction between the tissue powder and the buffer, promoting efficient lysis of the cells.

What is the purpose of incubating the sample on a thermal mixer after adding the lysis buffer?

Incubating the sample on a thermal mixer helps to facilitate the lysis process by providing a consistent temperature, which can enhance the breakdown of the plant cells and release of nucleic acids.

Why is it important to centrifuge the sample at maximum speed during the isolation process?

Centrifuging the sample at maximum speed helps to separate the soluble nucleic acids from the insoluble cell debris by creating a strong centrifugal force.

What should be done carefully when collecting the supernatant after centrifugation?

When collecting the supernatant, it is important to avoid aspirating the precipitated cell debris, as it may contain contaminants that could affect the quality of the nucleic acids.

Why is 95% ethanol added to the supernatant during the isolation process?

Adding 95% ethanol helps to precipitate the nucleic acids, allowing them to be separated from the remaining contaminants and facilitating their purification.

What is the role of the spin column in the nucleic acid isolation process?

The spin column is used to filter and purify the nucleic acids by allowing them to bind to a membrane while contaminants are washed away, resulting in a clean nucleic acid sample.