Plant RNA preparation experiment
Plant RNA preparation experiment
Since there are many sources of RNA species, there are different methods of extraction and preparation, generally phenol method, detergent method and guanidine hydrochloride method, of which phenol method is most commonly used in the laboratory. Tissue homogenization and centrifugation with phenol treatment, RNA is dissolved in the upper layer of the aqueous phase saturated with phenol, to the aqueous phase after the addition of cold ethanol, RNA that is precipitated as a white flocculent precipitate. This method is better to remove DNA and protein, and to obtain biologically active RNA.
Operation method
Phenol/SDS method
Materials and Instruments
RNA Move 1. Cool the mortar and rods with liquid nitrogen and weigh out 15 g of frozen plant tissue into the mortar (add liquid nitrogen to keep it frozen). For more product details, please visit Aladdin Scientific website.
TE LiCl anhydrous ethanol sodium acetate chloroform
Homogenizers Tubes Centrifuges Centrifuges Water Baths
2. Grind to a powder with the rod and immediately transfer to a 500 ml beaker containing 150 ml of Grinding Buffer and 50 ml of TLE Buffer Equilibrium Phenol.3. Homogenize for 2 min with a Poltroon homogenizer set at 5-6 stops, add 50 ml of chloroform and mix at low speed with the homogenizer.
4. Transfer the mixture to a 500 ml Nalgene centrifuge bottle and heat at 50°C for 20 min.
5. Centrifuge at 17,700 g for 20 min at 4°C with a JA-10 rotor.
6. Transfer the upper aqueous phase to another clean Nalgene centrifuge bottle (the interface is reserved for subsequent step 4), equilibrate the phenol with 50 ml of TLE buffer, mix well, and add 50 ml of chloroform.7. Transfer the residual water from the previous step together with the material at the interface into a 50 ml Oak Ridge centrifuge tube and centrifuge at 12,100 g with a JA-20 rotor for 20 min at 4°C.
8. Aspirate the aqueous phase, combine with the aqueous phase from step 3 and the mixture of phenol and chloroform, and mix vigorously.
9. Centrifuge at 17,700 g with a JA-10 rotor at 4 °C for 15 min and transfer the aqueous phase to another clean 500 ml centrifuge flask.
10. The aqueous phase was extracted repeatedly with a TLE equilibrium fraction until the interface between the two phases was clean, and the aqueous phase was extracted with chloroform.11. The aqueous phase was transferred into a 250 ml Nalgene centrifuge flask, LiCl was added to a final concentration of 2 mol/l (about 0.33 volume of 8 mol/l LiCl was added), and the precipitation was allowed to settle at 4°C overnight.
12. Centrifuge at 4°C with a JA-14 rotor 15 300 g for 20 min and the precipitate was washed with 2 to 3 ml, 2 mol/l LiCl solution.
13. The precipitate was redissolved in 5 ml of water and transferred to a 15 ml Sarstedt centrifuge tube, and the RNA was precipitated at 4°C for more than 2 h with the addition of 2 mol/l LiCl.
14. Centrifuge at 15,300 g with a JA-20 rotor at 4°C for 20 min, rinse with 2 mol/l LiCl solution, and redissolve in 2 ml of dry water.
15. Add 200 μl of 3 mol/l sodium acetate solution and 5.5 ml of anhydrous ethanol and precipitate at -20°C overnight or in dry ice/ethanol for 30 min.16. Centrifuge at 4°C with a JA-10 rotor at 17,700 g for 15 min, redissolve the precipitate in 1 ml of water and dilute 10 μl to 1 ml for A260 and A280.