Outlined below is the protocol that comes with ConcertPlant Reagent (Invitrogen). The protocol is not phenol-based, but requires the addition of chloroform. This reagent is used to isolate RNA from the tissues of a variety of plants, including blueleaf spruce needles, potato tubers, corn seeds, and cotton leaves. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

RNA purification experiments from plant tissues using ConcertPlant reagents

Materials and Instruments

Plant Tissue
Concert Plant RNA reagent NaCl Chloroform Isopropyl alcohol Ethanol DEPC treated water

Move

I. Materials

1. buffers, solutions and reagents

ConcertPlantRNA reagent (Invitrogen), pre-cooled at 4°C

NaCl, 5 mol/L (without RNAase)

Chloroform

Isopropyl alcohol

Ethanol, 75%, in DEPC-treated water

DEPC-treated water

2. Cells and tissues

Suitable plant tissues (see 1 in II. Methods), frozen in liquid nitrogen and ground fine

II. Methods

1. Add 0.5 ml of cold (4°C) Concert Plant RNA Reagent to no more than 0.lg of ground plant material. Mix with gentle shaking until the sample is resuspended in the reagent.

2. Place tubes horizontally at room temperature to warm for 5 min.

3. Centrifuge at 12000g for 2min to remove residue.

4. transfer the supernatant to a new tube without RNAase and add 0.1ml of 5mol/l NaCl. tap the tube on the lab bench to mix.

5. add 0.3 ml chloroform and turn the tube upside down several times to mix.

6. centrifuge at 12000g for l0min at 4°C.

7. Transfer the liquid phase to a new tube without RNAase. Add an equal volume of isopropanol to the liquid phase. Invert the tube several times to mix

Mix. Allow to incubate for 10 min at room temperature.

8. Centrifuge the sample at 12000 g for 10 min at 4°C.

9. Carefully remove and discard the supernatant to avoid disturbing the sediment.

10.Add lml of 75% ethanol to the sediment. Centrifuge the sample at 12000g for lmin at room temperature.

11. Dispose of the supernatant, taking care not to disturb the sediment. Centrifuge briefly and aspirate all residual liquid.

12. Redissolve the sediment in 10~30ul of DEPC treated water. If any cloudiness is seen, then at room temperature

Centrifuge the solution at 12000 g for 1 min. transfer the supernatant to a new RNAase-free tube and store at -70°C.

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