Plant DNA extraction - CTAB Method

Summary

This video demonstrates the step-by-step process of extracting DNA from plant samples using the CTAB method. It begins with the collection and grinding of plant material in liquid nitrogen, followed by lysis using CTAB buffer and incubation. The mixture is then centrifuged, and the supernatant is treated with chloroform and isoamyl alcohol for phase separation. The aqueous layer containing the DNA is collected, precipitated with ethanol and sodium chloride, and washed. Finally, the DNA pellet is resuspended in TE buffer and stored at 4°C for further analysis, highlighting the method's efficiency in obtaining pure DNA.

Takeaways

• 🌿 The video demonstrates the extraction of DNA from plant samples using the CTAB method.
• ⚗️ A 100 ml setup of CTAB lysis buffer is prepared for the extraction process.
• 🧪 Plant samples are collected, measured at 200 mg, and ground with liquid nitrogen for efficient cell disruption.
• 🔬 The ground samples are mixed with 700 µl of CTAB lysis buffer and vortexed to ensure thorough mixing.
• 🌡️ The mixture is incubated at 65°C for 20 minutes in a water bath to facilitate lysis.
• 🌀 After incubation, samples are centrifuged at 10,000 rpm for 10 minutes to separate the solid and liquid components.
• 🧊 The supernatant is transferred to a clean tube, and chloroform and isoamyl alcohol are added in equal volumes for phase separation.
• 🔄 The mixture is vortexed and centrifuged again to create an aqueous upper layer and an organic bottom layer.
• 🧫 The aqueous layer is carefully transferred to a new tube without disturbing the organic layer below.
• ❄️ Ice-cold ethanol and sodium chloride solution are added to precipitate the DNA from the aqueous layer.
• 🔍 Following centrifugation, a DNA pellet is observed at the bottom of the tube, which is then washed with 70% ethanol.
• 💨 The pellet is air-dried to remove residual ethanol before resuspension in TE buffer.
• 📦 Finally, the extracted DNA samples are stored at 4°C for future analysis.

Q & A

What is the main objective of the video?

The main objective of the video is to demonstrate the extraction of DNA from plant samples using the CTAB method.

What is the first step after collecting plant samples?

The first step after collecting plant samples is to measure 200 milligrams of the samples and grind them with liquid nitrogen.

What is the purpose of using liquid nitrogen in the process?

Liquid nitrogen is used to quickly freeze the plant samples, making them easier to grind into a fine powder without degradation.

What components are mixed with the ground plant samples in the lysis buffer?

The ground plant samples are mixed with 700 microliters of CTAB lysis buffer.

At what temperature and duration should the incubation occur after mixing with the lysis buffer?

The mixture should be incubated at 65 degrees Celsius for 20 minutes in a water bath.

What is the significance of centrifuging the samples at 10,000 rpm?

Centrifuging the samples at 10,000 rpm separates the cellular debris from the supernatant, allowing for the isolation of DNA.

What should be added to the supernatant after centrifugation, and why?

After centrifugation, an equal volume of chloroform and isoamyl alcohol should be added to help further purify the DNA by separating it from proteins and other contaminants.

What is the role of ice-cold ethanol in the DNA extraction process?

Ice-cold ethanol is used to precipitate the DNA from the aqueous layer, allowing it to form a visible pellet.

How is the DNA pellet washed after centrifugation?

The DNA pellet is washed with 600 microliters of 70% ethanol to remove any remaining impurities.

What should be done with the DNA pellet after washing?

After washing, the supernatant should be decanted, and the pellet should be air-dried before resuspending it in 50 microliters of TE buffer for storage.

At what temperature should the samples be stored after DNA extraction?

The samples should be stored at 4 degrees Celsius for further analysis.