RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)

Operation method

Isolation of RNA from Gram-positive bacteria

Materials and Instruments

10 ml Bacterial culture
DEPC-treated water Lysis buffer Phenol Chloroform Isoamyl alcohol 5 mol LNaCl Ice-cold anhydrous ethanol 70% ethanol DNA enzyme digestion buffer TE buffer
Centrifuge Ultrasonic machine with microprobe

Move

I. Materials and equipment

1) 10 ml of bacterial culture that

2)DEPC treated water.

3) Lysis buffer: 30 mmol/L Tris-Cl (PH7.4), 100 mmol/L NaCl, 5 mmol/L LEDTA, 1% (M/V) SDS, add Proteinase K to 100ug/ml before use.

4) Phenol: chloroform: isoamyl alcohol (25:24:1), phenol equilibrated with Tris-Cl (pH 8.0).

5) 24:1 chloroform:isoamyl alcohol

6) 5 mol/L NaCl.

7) Ice cold anhydrous ethanol and 70% ethanol.

8) DNA enzyme digestion buffer: 20 mmol/LTris-Cl (pH 8.0), 10 mmol/LMgCI2, 2.5 mg/ml DNA enzyme without RNAase

9) TE buffer (pH 8.0).

10) Centrifuge.

11) Ultrasonograph with microprobe.

II Methods of operation

1) Centrifuge at 12000 g at 4℃, recover cells from 10 ml of fine elaborated culture, resuspend the organisms in 0.5 ml lysis buffer and transfer to a microcentrifuge tube, freeze the plants on dry ice.

2) Thaw and sonicate with a microprobe sonicator at 30W for 3 times, l0S each time to avoid blistering, and incubate at 37℃ for 6Omin.

3) Add equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) extraction, centrifuge at high speed for 3 min at room temperature, and transfer the upper aqueous phase into another clean microcentrifuge tube.

4) Extract with phenol: chloroform: isoamyl alcohol (25:24:1) once more, and then extract with chloroform: isoamyl alcohol (24:1) once more.

5) Add 15ul of 5mol/L NaCl to 400ul of aqueous phase and fill with ice-cold anhydrous ethanol, mix well and leave on ice for 15~30 min or overnight at -20℃.

6) Centrifuge at 4℃ for 15 min, rinse the precipitate with ice-cold 70% ethanol, air dry, dissolve the precipitate with 95ul DMA enzyme digestion buffer, add 4ul 2.5 mg/ml DNA enzyme I without RNAase, incubate at 37℃ for 60 min.

7) Extract with phenol: chloroform: isoamyl alcohol (25:24:1) once, remove the aqueous phase, add 100ul of TE rinse solution to the organic phase, mix well, and centrifuge at high speed for 5min at room temperature in a microhalo centrifuge, and then combine the two aqueous phases.

8) Extract the aqueous phase once with chloroform:isoamyl alcohol (24:1), add 10ul of 5mol/L NaCl and 60ul of anhydrous ethanol, overnight at 20°C or put it in dry ice/ethanol for 15 min, and then centrifuge it at high speed in a microcentrifuge at 4°C for 15~30 min.

9) Wash the precipitate with 500ul of 70% ethanol and air dry. Dissolve in 100ul of DEPC-treated water. Dissolve in 100ul of DEPC-treated water. Dilute 10ul of DEPC-treated water in lml of water. Measure _A260 and _A280 to quantify RNA, store at -70℃ or store as ethanol precipitate.

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