Influenza virus RNA extraction experimental steps - Database & Sql Blog Articles

Influenza virus RNA extraction experimental procedure, a material and equipment 1. Phenol extraction method 1) 10 × RSB solution: O.Olmol / LTris, O.OOlmol / L KCl, 0.0015mol / LMgCL22) 10% SDS3) Proteinase K, 5 mg / Ml4) lOXLiCI solution: 5% SDS, 0.1 mol/L sodium acetate, 1.4 mol/L LiCl. 5) RSB saturated phenol. 6): isoamyl alcohol (24:1) 7) 2 mol/L sodium acetate. 8) Anhydrous ethanol. 2. LiCl extraction method 1) TSE solution. 2) 10% SDS, 3) Proteinase K (10 mg/ml). 4) lOXLiCl solution: 5% SDS, 0.1 mol/L sodium acetate, 1.4 mol/L LiCl5) LiCl buffer to balance phenol 6) 7) 2 mol/L sodium acetate 8) absolute ethanol, 3) phenol-SDS extraction method 1 ) TSE solution 2) 10% SDS3) TSE saturated phenol 4) 4 mol/L LiCL 5) 2 mol/L sodium acetate. 6) Anhydrous ethanol. Influenza virus RNA extraction experimental procedure, two methods of operation 1. Phenol extraction 1) Add 1/10 volume of 1OXRSB, 0.12 ml of 10% SDS, 0,4 ml of proteinase K to 4 ml of purified virus suspension, set at 56 ° C for 15 min 2) Add 0.8 ml of OXLiCl solution and 4 ml of phenol saturated with RSB to the above solution, shake at 56 ° C for 5 min, and immediately ice bath. 3) Add 4 ml: isoamyl alcohol (24:1), shake at room temperature for 15 min, 3000 r/min, and centrifuge for 10 min. 4) Take the upper aqueous phase, add 4 ml of RSB saturated phenol and 4 ml: isoamyl alcohol, repeat 1~2 times. 5) Take the upper aqueous phase, add 0.1 volume of 2mol/L sodium acetate, 2.5 times the volume of no Water ethanol, stored at -20 ° C or -70 ° C. 2. LiCl extraction method 1) The purified virus was resuspended in 2 ml of TSE, 70ul 10% SDS (final concentration 0.3%), and 100ul proteinase K (10 mg/ml) was added and placed at 56 ° C for 15 min. 2) Add 0.2 ml of 10XLiCl buffer, 1 ml of phenol solution equilibrated with LiCl buffer, gently rub, centrifuge at 2000r/mm for 5 min3) Take the upper phenol phase, add 0.5 ml of 1XLiCl solution, extract once, centrifuge at 2000r/min for 5 min4) The upper aqueous phase was mixed with the lower aqueous phase obtained in 3), and 1.2 ml of LiCl-balanced phenol was added, and the mixture was gently shaken at 2000 r/min for 5 min. 5) Discard the upper phenol phase, add 1.2 ml, gently rub, and centrifuge at 5 μm at 2000 r/min. 6) Take the upper aqueous phase, add 0.05 times volume of 2mol/L sodium acetate volume of absolute ethanol, store at -20 °C or -70 °C 3. Phenol-SDS extraction method 1) Resuspend the purified virus in 2 ml TSE Add 0.2 ml of 10% SDS and gently shake until the suspension is clear. 2) Add 2 ml of TSE saturated phenol, shake gently for 5 min in an ice bath, and centrifuge at 3000 r/mim for 10 min. 3) Take the upper aqueous phase and extract it 2 to 3 times with TSE saturated phenol. 4) Take the upper aqueous phase, add 100ul 4mol/L LiCl, 0.05 times volume 2mol/L sodium acetate, 2.5 times volume absolute ethanol, and store at -20°C or -70°C.