Large-scale extraction and purification of plasmid DNA and genetic transformation of cotton
Experimental reagent
1. STE [0.1 mol/L NaCI, 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/LEDTA (pH 8.0)]
2. Solution I [50 mmol/L sucrose, 25 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA (pH 8.0)]
3. Solution II (mixing 0.4 mol/L NaOH and 2% SDS)
4. Solution III (3 mol/L Kac, pH 4.8)
5. Isopropanol
6. 4 mol/L LiCI
7. RNase
8. Phenol, phenol-chloroform, chloroform
9. Ethanol
Laboratory equipment
Shaker
2. Ice machine
3. High speed centrifuge
4. Water bath
5. Ultra-clean workbench
6. Glass capillary
7. Microinjector
Experimental Materials
Cotton, transformed Agrobacterium strain
Experimental procedure
1. Shake 500 ml at 37 ° C, 250 rpm.
2. Centrifuge at 5000 × g for 5 min to collect the cells, and wash the cells once with 50 ml of STE.
3. Add 20 ml of the suspension to the solution I.
4. Add solution 40 ml, gently invert the tube several times and place in an ice bath for 20 min.
5. Add 30 ml of pre-cooled solution, invert the tube several times, and place in an ice bath for more than 10 min.
6. Centrifuge at 10000 × g for 15 mm, add 0.6 times volume of isopropanol to the supernatant, mix well and leave at room temperature for more than 10 min.
7. Centrifuge at 10,000 × g for 10 min and discard the supernatant. After the precipitate is dried, dissolve it with 350 ul of water, add 350 ul of 4 mol/L LiCI, mix and leave at room temperature for more than 10 min.
8. Centrifuge at 10000 × g for 5 min, add the supernatant to RNase to a final concentration of 100 ug/ml, and incubate at 37 °C for 2 h.
9. Extract once with phenol, phenol-chloroform, and chloroform. The supernatant was added with 0.1 volume of 3 mol/L NaAc (pH 5.2) and 2 volumes of absolute ethanol.
10. Collect the DNA pellet by centrifugation and wash once with 70% ethanol. After drying, dissolve in TE and set aside.
11. Genetic transformation of cotton: A large number of plasmids were diluted with sterile water to a 2 mg/ml DNA solution for ovary injection. The injection was carried out on a clean bench, which was a glass capillary with a tip diameter of approximately 0.1 mm. The other end of the capillary was tightly fitted into a 10-20 ul micro syringe needle and 6-12 ul of the injected DNA sample was aspirated. When injecting, the needle is directed at the portion of the filament that protrudes from the ovary. Each ovary was injected with approximately 0.2 ul of DNA.
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