Under liquid nitrogen conditions, cells of polyphenol-rich plant leaves are broken down using physical grinding methods. Antioxidants are used to prevent the oxidation and browning of polyphenolic compounds. This method is suitable for extracting and purifying plant genomic DNA from tea plants, potatoes, cherries, grapes, and other polyphenol-rich plants.
**Equipment Used**
1. Cryogenic Grinder (samples are frozen and ground under liquid nitrogen conditions)
2. Centrifuge (capable of centrifugation at 4℃ with speeds above 10,000 rpm)
3. Water Bath (operating range of 60℃~70℃)
4. Vortex Mixer
5. Horizontal Electrophoresis Apparatus
6. Gel Imaging System
7. Adjustable Pipettes
8. UV Spectrophotometer
9. Balance (accuracy of 0.1 mg)
**Analysis Steps**
1. Take fresh young leaves of the plant and rinse them with sterile ultrapure water for 1 minute. Disinfect with 70% ethanol for 2 minutes, then rinse with sterile ultrapure water for 2 minutes. Dry the leaves with sterile absorbent paper and weigh approximately 0.5g (accurate to 0.1mg).
2. Place the clean sample in a sterilized grinding vial and use the grinder to grind the sample into a powder. Transfer the ground sample with a sterile glass rod using a Mechanical Pipette into a pre-cooled 15ml centrifuge tube containing 5ml DNA extraction buffer. Add 0.09g Na2S2O5, 0.2g PVP, and 500µl of ethanol, invert to mix thoroughly. Place the centrifuge tube in the H1750R refrigerated microcentrifuge and centrifuge at 5000 r/min for 5 minutes at 4℃.
3. Remove the supernatant and add 5 ml of preheated (65℃) DNA extraction buffer. Gently invert to mix, then incubate in an LCH-WB-4 general water bath at 65℃ for 1 hour, shaking every 10 minutes.
4. Cool the centrifuge tube on ice and add 1 ml of pre-cooled NaAc solution.
5. Mix well and add 5ml of chloroform: isoamyl alcohol: ethanol solution. Gently invert to mix (wear gloves to protect the skin), and let stand on ice for 5-10 minutes to allow phase separation (re-mix if necessary). Centrifuge at 4℃ and 10000 r/min for 10 minutes, transfer the supernatant to a sterile 15ml centrifuge tube and repeat this step.
6. Transfer the flocculent DNA precipitate to a sterile centrifuge tube containing 600µl of TE buffer.
7. Add RNaseA solution to a final concentration of 10µg/ml, incubate at 37℃ for 30 minutes, add 0.1g PVP, and add an equal volume of chloroform: isoamyl alcohol: ethanol solution. Gently invert to mix, then centrifuge at 10000 r/min for 10 minutes.
8. Transfer the supernatant to a sterile 1.5ml centrifuge tube, perform one more extraction, add 1/10 volume of NaAc solution, and add two volumes of pre-cooled absolute ethanol. Mix well and place at -20℃ for about 20 minutes.
9. Centrifuge at 4℃ and 10000 r/min for 10 minutes, discard the supernatant, add 1 ml of 70% ethanol, and gently invert the centrifuge tube several times to wash the pellet. Repeat this step twice.
10. Add 500µl of pre-cooled absolute ethanol to the pellet and centrifuge at 4℃ and 10000 r/min for 5-8 seconds.
11. Discard the ethanol solution, leave the centrifuge tube open at room temperature to allow ethanol to evaporate, and then dissolve the DNA in 200µl of TE buffer.
**Precautions**
1. ensure adherence to the specified temperature and speed when using the centrifuge.
2. When preparing samples, completely dry the moisture and measure the weight accurately to 0.1mg.
3. Follow proper usage protocols when handling the equipment.
