Extraction of mitochondrial DNA from plant cells

In eukaryotes, in addition to the nuclear genome, there are cytoplasmic genomes, i.e., the mitochondrial genome and, in green plants, the chloroplast genome. Both the mitochondrial genome and the chloroplast genome are cyclic molecules, and their size varies with species. Mitochondrial DNA (mtDNA) ranges in size from 200 to 2500 kb. The organelle genome is different from the nuclear genome in terms of mode of inheritance, genome size and structure, gene coding capacity, gene expression and regulation, etc., and the chloroplast and mitochondrial genomes each have their own characteristics. The mutual coordination of nuclear and plasmid genomes in function is an important basis for the cell to carry out life activities. Therefore, it is of great significance to study the structure and function of organelle genomes. In all studies of organelle genomes, the isolation of purified organelle DNA is extremely critical.

Operation method

basic program

Principle

The principles of separating mitochondrial DNA and chloroplast DNA are basically the same. The method starts with the isolation of intact organelles and then the extraction of DNA from the organelles.To obtain high purity organelle DNA, it is crucial to separate the desired organelle from other subcellular structures, which can be accomplished by differential or gradient centrifugation.After lysis of the intact organelle, DNA can be obtained by CsCl centrifugation or phenol-chloroform extraction. After the complete organelles were lysed, the DNA could be obtained by CsCl centrifugation or phenol-chloroform extraction, and DNase was often used to clear the DNA from the non-organelles before lysing the organelles. mitochondria were separated from the cells by homogenization, and then the mitochondria were lysed, which released the DNA and proteins, and then phenol was used to extract the purified mtDNA.

Materials and Instruments

Young plant leaves
Mortar (12 cm diameter) and rods Freezing centrifuge (Sorvall Beckman et al.) Microcentrifuge (1.5 ml tubes) -20°C refrigerator Constant temperature water baths

Move

All the following operations were carried out at 4°C unless otherwise indicated, with the mortar, rod and centrifuge tube pre-cooled, using chilled buffer and operating in an ice bucket.

1. Young leaves were cut, sterilized with 1% sodium hypochlorite solution for 15 min, rinsed three times with sterile water, and then cut into pieces of about 1 ^cm2.

2. Add grinding buffer at the ratio of 10 ml of grinding buffer per gram of material, and grind the leaves into homogenate in a mortar. The filtrate was filtered through 6 layers of gauze and collected.

3. The filtrate was centrifuged at 3,000 r/min for 15 min at 4°C, and the supernatant was collected.

4. The supernatant was centrifuged at 4℃, 10000r/min for 25min, and the supernatant was discarded.

5. Suspend the precipitate with buffer A (without adding β-mercaptoethanol, BSA and PVP), and repeat the steps 4 and 5, and the collected precipitate is the crude mitochondria.

6. Add MgCl₂ to a final concentration of 5 mmol/L, add DNase Ⅰ to a final concentration of 30 μg/ml, add _Na2EDTA to a final concentration of 15 mmol/L after 1h ice bath to terminate the DNA digestion reaction.

7. Spread the crude mitochondria on a discontinuous concentration of sucrose gradient (sucrose concentration from top to bottom was 20%, 40%, 52%, 60%, the volume was 7ml, 10ml, 10ml, 7ml, prepared by buffer C). 4℃, 20,000r/min ultracentrifugation for 2.5h, and aspirate mitochondria at the interface of 40% and 52% sucrose.

8. In the mitochondrial aspirate add 4 times the volume of buffer B. 4 ℃, 10,000 r/min centrifugation for 15 min. the resulting precipitate is pure, intact mitochondria without nuclear DNA contamination on the surface.

9. Suspend the mitochondria in lysis buffer, add 1/10 volume of 10% SDS and 1/100 volume of 30mg/ml RNase solution, 50 ℃ water bath for 30min.

10. Add 1/150 volume of 25mg/ml proteinase K, 37℃ water bath for 30min.

11. Extract DNA with phenol, phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform sequentially.

12. Add 1/10 volume of 3 mol/L sodium acetate and 2 times the volume of anhydrous ethanol, mix well, and leave at -20℃ for at least 30min, then centrifuge at 4℃ for 15min at 13000r/min in a microcentrifuge to collect DNA.

13. The precipitated DNA was washed with 75% ethanol for 2~3 times.

14. The DNA precipitate was dried in air and dissolved in a small amount of TE buffer (10 mmol/LTris-HCl, 1 _{mmol/LNa2EDTA}, pH 8.0). Store at -20℃ for spare parts.

Caveat

Plants used for extracting mitochondrial DNA are preferably grown under dark conditions to obtain yellowed seedlings to inhibit chloroplast development and minimize chloroplast interference when isolating mitochondria

Common Problems

Specific reagent description:

Buffer A (grinding buffer) (0.4 mol/L mannitol, 50 mmol/L Tris-HCl, 1 mmol/L _Na2EDTA, 5 mmol/L KCl, pH 7.5; 2 mmol/L β-mercaptoethanol, 0.1% BSA, 10 mg/ml poly(vinylpyrrolidone) was added before use), Buffer B (0.2 mol/L mannitol, 10 mmol/L Tris-HCl, 1 mmol/L _Na2EDTA, pH 7.2), buffer C (1 mmol/L _Na2EDTA, 15 mmol/L Tris-HCl, pH 7.2), proteinase K (25 mg/ml), sucrose (20%, 40%, 52%, 60%), MgCl₂ (0.1 mol/L), DNaseI (2 mg/ml, dissolved in water), TE buffer (10 mmol/L Tris-HCl, 1 mmol/L _Na2EDTA, pH 8.0), SDS (10%), RNase (30 mg/ml), sodium acetate (3 mol/L), ethanol, phenol, chloroform, isoamyl alcohol.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/