Isolation of Yeast RNA

Summary

This video provides a step-by-step guide for isolating RNA from yeast. The process begins by preparing a mixture of sodium hydroxide solution and water, followed by heating and occasional stirring. After straining the mixture, it undergoes multiple centrifugation and acidification steps, using glacial acetic acid to adjust pH. The solution is then evaporated, centrifuged again, and treated with ethanol and hydrochloric acid to isolate the RNA. The result is a purified yeast RNA sample, ready for further analysis or experimentation.

Takeaways

• 😀 The experiment starts with the preparation of yeast isolation using 3 grams of dry yeast.
• 😀 A 1% sodium hydroxide solution (5 ml) is used in the initial step of the process.
• 😀 25 ml of water is added to the sodium hydroxide solution and the mixture is heated in a water bath for 15 minutes.
• 😀 During the 15-minute heating process, occasional stirring is required to mix the solution.
• 😀 After heating, the solution is strained through a cheesecloth to collect the filtrate.
• 😀 The filtrate is centrifuged to separate the components, ensuring the centrifuge is balanced properly.
• 😀 After centrifugation, the supernatant is transferred to a beaker.
• 😀 Glacial acetic acid is added dropwise to the supernatant until it becomes faintly acidic, checked using blue litmus paper.
• 😀 The solution becomes turbid, requiring a second round of centrifugation.
• 😀 The supernatant is evaporated over a water bath to reduce its volume to approximately 5 ml.
• 😀 A third centrifugation is conducted, followed by the addition of 10 ml of 95% ethanol and 3 drops of concentrated hydrochloric acid.
• 😀 The final result of the process is the isolation of RNA from the yeast.

Q & A

What is the first step in isolating RNA from yeast in this procedure?

The first step involves preparing the required reagents: 3 grams of dry yeast, 5 mL of 1% sodium hydroxide solution, and 25 mL of water.

Why is the sodium hydroxide solution mixed with water before heating?

The sodium hydroxide solution is mixed with water to create an alkaline environment that helps break down the yeast cells and release their RNA.

How long should the mixture be heated in the water bath?

The mixture should be heated in the water bath for 15 minutes, with occasional stirring to ensure proper mixing.

What role does the cheesecloth play in the RNA isolation procedure?

The cheesecloth is used to strain the mixture, allowing the filtrate to be collected while separating out any solid particles from the liquid.

What is the purpose of centrifuging the filtrate after straining?

Centrifugation helps separate the solid particles (pellet) from the liquid (supernatant), which contains the RNA, by using centrifugal force.

How can you determine if the supernatant is acidic enough after adding glacial acetic acid?

The acidity of the supernatant can be tested using blue litmus paper. Once the litmus paper turns red, it indicates the solution is faintly acidic.

Why is a second centrifugation needed after acidification?

A second centrifugation is needed to remove any remaining turbidity (particles) in the solution, ensuring a cleaner supernatant.

What is the purpose of evaporating the supernatant in a water bath?

The supernatant is evaporated to concentrate the solution, removing excess liquid and making the RNA more concentrated for further processing.

How does adding ethanol assist in isolating RNA?

Ethanol is added to precipitate the RNA out of the solution, helping to separate the RNA from other components in the liquid.

What does the final addition of concentrated hydrochloric acid achieve in the process?

The addition of concentrated hydrochloric acid ensures that the RNA is fully precipitated and ready for further analysis or use in experiments.