Small Amounts of DNA from Novel Plant Genomes Extraction Kit

1. Components of the reagent kit

2. Storage

This kit should be stored at room temperature (15-25℃) in dry conditions and can be stored for 12 months. DNA extraction purification columns can be stored in a cool and dry environment for up to 1 year. RNase A contains a preservative and can be transported at room temperature, but for long-term storage, it should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, liquid nitrogen, chloroform, sterile deionized water, and EP tubes.

4. Introduction to the Reagent Kit

The new plant genome DNA purification kit provides a rapid method for DNA purification. It effectively precipitates DNA with specific reagent buffers II and C, collecting high-purity DNA through adsorption columns.

This kit is widely applicable for isolating samples from plant tissues and fungi, enabling the extraction of total DNA from plants and fungi within 30 minuta. The extraction process doesn’t involve toxic reagents like phenol-chloroform. The extracted DNA can be directly used for downstream experiments such as PCR and Southern blotting.

5. Experimental Principles and Procedures：

6. Extraction Process

Precautions before starting the experiment:

A. Reagent Buffer I and Reagent Buffer C tend to precipitate at low temperatures. It is recommended to heat them at 65°C for 5 minutes until the precipitates dissolve before normal usage.

B. Before using Wash Buffer 1, add the specified amount of absolute ethanol as indicated on the reagent bottle label and mark a check (√) on the label to indicate the addition of absolute ethanol.

C. The Elution Buffer is a 0.1x TE solution containing a minimal amount of EDTA. If EDTA affects subsequent experiments, it is recommended to substitute the Elution Buffer with sterile deionized water.

1. Sample Processing:

A. Material Collection and Storage

If freshly collected materials cannot be immediately used, place them in liquid nitrogen for cooling and finally store at -80°C. Dried materials can be stored at room temperature.

B. If conditions permit, collect fresh materials whenever possible, as fresh materials contain fewer polysaccharides and polyphenols.

C. When collecting fungi from liquid culture, separate the liquid and collect the fungal cells by centrifugation.

2. Weigh around 100 mg of fresh samples or not exceeding 20 mg of dry material and grind it with liquid nitrogen.

(Note: Different sample amounts may vary; it is advisable to optimize the sample amount through pre-experimental trials.)

3. Dodati 400μl Reagent Buffer I and 10μl RNaseA (10 mg/ml), ensuring there are no clumps in the ground sample. Clumps are difficult to lyse, reducing the DNA yield. Also, do not mix Reagent Buffer I and RNaseA before usage.

4. Incubate at 65°C for 5 minuta, gently invert the mixture 2-3 times. This step is used for cell lysis. If samples are difficult to lyse, extend the incubation time, but not beyond 30 minuta.

5. Dodati 130μl Reagent Buffer II and mix well, then ice-bathe for 5 minuta (this step is used for precipitating polysaccharides and proteins).

6. Centrifuge the lysate for 5 minutes at 14,000 rpm (20,000×g).

(Note: Some plant materials may contain a lot of viscous substances at this step, which can shear DNA in the subsequent steps. Therefore, the ideal state is to remove these substances during this step. After centrifugation, transfer the supernatant to a new centrifuge tube. If there is a significant amount of flocculent material in the supernatant after centrifugation, it indicates that the initial sample quantity was too large. Consider reducing the initial sample amount.)

7. Carefully transfer the obtained liquid to a new centrifuge tube.

(Note: Approximately 450μl of liquid can be transferred; for some species, it may be less than 450μl.)

8. Add an equal volume of Reagent Buffer C and an equal volume of absolute ethanol to the lysate and mix well.

(For example: Add 450μl of Reagent Buffer C, then add 450μl of absolute ethanol. If the volume of the lysate is less than 450μl, reduce the amount of Reagent Buffer C proportionally. Adding Reagent Buffer C will cause slight precipitation, but it won’t affect subsequent experiments.)

9. Add the obtained liquid to the DNA extraction purification column (kit) (approximately 650-700μl each time), centrifuge at greater than 8,000 rpm for 1 minute, discard the collected waste, and re-insert the collection tube into the purification column for the next step.

10. Repeat step 9, add the remaining liquid to the DNA extraction purification column (kit), centrifuge at greater than 8,000 rpm for 1 minute, discard waste and the collection tube.

11. Place the DNA extraction purification column (kit) into the collection tube, add 300μl of Wash Buffer 1,centrifuge at greater than 8,000 rpm for 1 minute, discard waste, and place the DNA extraction purification column (kit) back into the tube for the next step.

(Note: Confirm the addition of absolute ethanol in Wash Buffer 1.)

12. Dodati 500μl of Wash Buffer 1 to the DNA extraction purification column (kit), centrifuge at 14,000 rpm (20,000×g) za 2 minuta, extend centrifugation time appropriately to ensure the membrane is adequately dry.

13. Place the DNA extraction purification column (kit) into a new centrifuge tube, leave it uncovered, and incubate at 65°C for 2 minuta. Extend this step as needed to evaporate ethanol to prevent ethanol residue from affecting downstream experiments.

14. Pipeta 100μl of Elution Buffer onto the column membrane, centrifuge at 12,000 rpm for 2 minuta.

(Note: 1. Using 50μl of Elution Buffer to elute DNA can increase DNA concentration but reduce the overall DNA yield; 2. The eluate containing DNA can be reapplied to the DNA extraction purification column and centrifuged at 12,000 rpm for 2 minutes again to increase DNA yield.)