Nucleic Acid Isolation from Plant Soft Tissues
Résumé
TLDRThe video provides a detailed protocol for isolating nucleic acids from plant tissues, emphasizing the importance of using liquid nitrogen to cool grinding equipment and samples. It outlines the process of grinding plant material into a fine powder, preparing a lysis buffer, and subsequent steps, including incubation, centrifugation, and adding ethanol to precipitate nucleic acids. The extracted nucleic acids can be purified using a spin column or magnetic beads. Critical safety and procedural tips are highlighted throughout to ensure successful isolation.
A retenir
- 🌿 Ensure grinding equipment and tubes are pre-cooled with liquid nitrogen.
- 🧊 Place soft plant tissues in liquid nitrogen before grinding.
- 🔬 Grind tissues into a fine powder for effective processing.
- ❄️ Store powdered samples in liquid nitrogen or at -70°C for preservation.
- ⚗️ Prepare and weigh the lysis buffer before sample addition.
- 🌀 Vortex immediately after adding plant powder to ensure mixing.
- 🔥 Incubate the lysed sample on a thermal mixer for proper cell lysis.
- ⚖️ Centrifuge at maximum speed to separate cell debris from supernatant.
- 💧 Carefully collect the supernatant, avoiding aspirating debris.
- 🍃 Use 95% ethanol to precipitate and purify nucleic acids.
Chronologie
- 00:00:00 - 00:02:05
The protocol for plant nucleic acid isolation from soft tissues begins with ensuring all grinding equipment and tubes are cooled with liquid nitrogen. Next, cut the soft tissues from the selected plant material and immerse them in liquid nitrogen. Sufficient liquid nitrogen is required for effective grinding. The tissue is ground into a fine powder using a pestle, and the powdered sample is either stored in liquid nitrogen or at -70 degrees Celsius. The lysis buffer preparation includes weighing the lysis tube with the buffer, then adding up to 50mg of plant powder to it. After adding the powder to the buffer, mix and lyse the sample using a thermal mixer, then centrifuge at maximum speed. Carefully collect the supernatant without disturbing the precipitated debris and transfer it to a new tube. Add 95% ethanol and mix thoroughly. The sample can be applied to a spin column or purified using a magnetic bead protocol.
Carte mentale
Vidéo Q&R
What is the purpose of cooling equipment with liquid nitrogen?
Cooling equipment with liquid nitrogen prevents sample degradation by keeping it at a low temperature during grinding.
Why is it important to grind plant tissues into a fine powder?
Grinding into a fine powder increases the surface area for better extraction and improves the efficiency of nucleic acid isolation.
How should the powdered sample be stored?
Powdered samples should be stored in liquid nitrogen or at -70°C to preserve nucleic acid integrity.
What is the role of the lysis buffer in nucleic acid isolation?
The lysis buffer helps break down cell walls and membranes, releasing nucleic acids into solution for extraction.
How is the supernatant collected?
After centrifugation, the supernatant is collected carefully to avoid aspirating any precipitated debris.
Why is ethanol added to the supernatant?
Ethanol precipitates nucleic acids out of the solution, allowing them to be separated from other cellular components.
Is there an alternative to using a spin column?
Yes, magnetic bead purification can be used as an alternative method for nucleic acid purification.
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- plant nucleic acid isolation
- soft tissues
- liquid nitrogen
- grinding
- lysis buffer
- centrifuge
- supernatant
- ethanol
- spin column
- magnetic bead purification