RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Influenza virus RNA extraction
Materials and Instruments
10× RSB solution 10% SDS Protease K lOXLiCI solution RSB Saturated phenol Chloroform Isoamyl alcohol 2mol L Sodium acetate Anhydrous ethanol TSE solution LiCl buffer equilibrium Phenol TSE Saturated phenol 4mol LLiCL Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
1. Phenol extraction
1) 10×RSB solution: O.Olmol/LTris, O.OOlmol/LKCl,0.0015mol/LMgCL2
2)10%SDS
3) Protease K, 5 mg/ml
4) lOXLiCI solution: 5% SDS, 0.lmol/L sodium acetate, 1.4 mol/L LiCl.
5) RSB saturated phenol .
6) Chloroform: isoamyl alcohol (24:1)
7) 2mol/L sodium acetate.
8) Anhydrous ethanol.
2. LiCl extraction
1) TSE solution.
2) 10% SDS.
3) Protease K (10 mg/ml).
4) lOXLiCl solution: 5% SDS, 0.lmol/L sodium acetate, 1.4 mol/L LiCl
5) LiCl buffer equilibrium phenol
6) Chloroform
7) 2mol/L sodium acetate
8) anhydrous ethanol.
3) Phenol-SDS extraction
1) TSE solution
2) 10% SDS
3)TSE saturated phenol
4)4mol/LLiCL
5)2mol/L sodium acetate.
6) Anhydrous ethanol.
II Methods of operation
1. Phenol extraction
1) Add 1/10 volume of 1OXRSB, 0.12 ml of 10% SDS, 0.4 ml of protease K to 4 ml of purified virus suspension, and set at 56℃ for 15 min.
2) Add 0.8 mllOXLiCl 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 of chloroform:isoamyl alcohol (24:1), shake at room temperature for 15 min, centrifuge at 3000 r/min for 10 min.
4) Take the upper layer of aqueous phase, add 4 ml RSB saturated phenol and 4 ml chloroform: isoamyl alcohol, and so on for 1 ~ 2 times.
5) Take the upper aqueous phase, add 0.1 times the volume of 2mol/L sodium acetate, 2.5 times the volume of anhydrous ethanol, -20 ℃ or -70 ℃ storage.
2.LiCl extraction method
1) Suspend the purified virus in 2 ml of TSE, add 70ul of 10% SDS (final concentration of 0.3%), add 100ul of Proteinase K (10 mg/ml), and set at 56℃ for 15 min.
2) Add 0.2 ml of 10XLiCl buffer, 1 ml of phenol solution equilibrated with LiCl buffer, lightly moisten, centrifuge at 2000r/mm for 5 min.
3) take the upper layer of the phenol phase, add 0.5 ml1XLiCl solution, extract once, centrifuge at 2000r/min for 5 min.
4) Take the upper aqueous phase and mix it with the lower aqueous phase obtained in 3), then add 1.2 mlLiCl equilibrated phenol, shake gently, centrifuge at 2000r/min for 5 min.
5) Discard the upper phenol phase, add 1.2 ml of chloroform, shake gently and centrifuge at 2000 r/min for 5 mim.
6) Take the upper aqueous phase, add 0.05 times the volume of 2mol/L sodium acetate times the volume of anhydrous ethanol, and store at -20°C or -70°C.
3. Phenol-SDS extraction
1) Resuspend the purified virus in 2 ml of TSE, add 0.2 ml of 10% SDS and shake gently until the suspension is clear.
2) Add 2 ml TSE saturated phenol, shake gently in an ice bath for 5 min, 3000r/mim centrifugation lOmin.
3) Take the upper aqueous phase, and then extract with TSE saturated phenol 2~3 times.
4) Take the upper aqueous phase, add 100ul of 4mol/L LiCl, 0.05 times the volume of 2mol/L sodium acetate, 2.5 times the volume of anhydrous ethanol, and store at -20°C or -70°C.