Grind the plant material in liquid nitrogen, make some cells broken, further make the plant cells lysed in lysis solution, precipitate protein with sodium acetate and chloroform, precipitate nucleic acid with isopropanol, dissolve and precipitate total RNA with lithium chloride, and then wash to get high quality RNA.The purpose of this experiment is to understand and grasp the method of isolation and purification of total RNA from plants.

Operation method

abrasive method

Principle

Grind the plant material in liquid nitrogen, make some cells broken, further make the plant cells lysed in lysis solution, precipitate protein with sodium acetate and chloroform, precipitate nucleic acid with isopropanol, dissolve and precipitate total RNA with lithium chloride, and then wash to get high quality RNA.The purpose of this experiment is to understand and grasp the method of isolation and purification of total RNA from plants.

Materials and Instruments

Plant RNA
Urea Tris-HCl Na2 EDTA NaCl Tris saturated phenol SDS LDS sodium acetate chloroform-isoamyl alcohol isopropyl alcohol TE buffer LiCl ethanol
Mortar Beaker Centrifuge

Move

Take 5-10 g of the material into a mortar, add excess liquid nitrogen, and quickly grind it into a homogeneous powder (ensure that the material is always immersed in liquid nitrogen during the grinding process, and grind it for about 30 min). After the liquid nitrogen evaporated naturally, transfer the material into a 100 ml centrifuge tube, add 6-8 times the volume of lysate1 , stir gently, and then centrifuge at 12000 r/min for 15 min at 4 ℃ for 20 min in an ice bath at 0 ℃. Take the supernatant, add 1/3 of the volume of 3 mol/ L sodium acetate, 1/5 of chloroform isoamyl alcohol, shake gently, 0 ℃ ice bath for 20 min. 2000 r/ min, 4 ℃ ice bath. Centrifuge at 2000 r/min for 15 min at 4 ℃ (add 5 times volume of lysis solution2 to dissolve the precipitate for DNA extraction). Take the supernatant, add an equal volume of ice-cooled isopropanol, mix well and ice bath for 10 min. After centrifugation at 5000 r/min for 10 min at 4 ℃, the precipitate was dissolved in 5 ml of TE buffer containing 0.1% SDS. The precipitate was dissolved in 5 ml of TE buffer containing 0.1% SDS, and 8 mol/ L LiCl was added to reach a final concentration of 2.5 mol/ L. The solution was then mixed in ice bath. Add 8 mol/L LiCl to reach a final concentration of 2.5 mol/L, and incubate for 3 h in an ice bath or overnight at 4 ℃. The precipitate was centrifuged at 12000 r/min for 10 min at 4 ℃.
The precipitate was washed twice with 70% ethanol, dried in air for 10 min, dissolved in sterile water (DEPC diethyl ether pyrocarbonate, treated), added 1/10 of 10% by volume of SDS, and repeated the steps 4-6 to further remove proteins, and stored in 70% ethanol at 20 ℃ for a long time.

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