RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Encephalitis B virus RNA extraction
Materials and Instruments
Phenol Chloroform Isoamyl alcohol Ether KAc Ethanol DEPC Treated water TBE Buffer Sampling solution KCI. Move I. Materials and equipment Caveat 1) In order to obtain a high titer of virus, it is necessary to select cultured cells that are sensitive to encephalitis B virus. Pair c6/36 cultured cells have a high titer of virus propagation, and the R virus is able to cause continuous infection of the cells with minimal cellular lesions. After virus infection, the virus can be harvested after 2 to 4 days of incubation, and the culture can be continued by replacing the culture medium with fresh culture medium, so that the virus-containing supernatant can be obtained continuously from a batch of cultured cells.2) The purified virus RNA is stable in ethanol at -20℃ and can be stored for at least 2 months. For more product details, please visit Aladdin Scientific website.
Dialysis bag
1) Phenol: Chloroform: Isoamyl alcohol (25:24:1),
2) Ether
3) 2.5mol/LKAc
4) Ethanol
5) DEPC treated water
6) TBE buffer
7] Sample solution: 40% glycerol, 0.4% TBE solution of brown tinted bromine red
S) Dialysis bag
9) KCI
II Methods of Operation
1. Phenol: chloroform: isoamyl leaven extraction method
1) Purify the virus by centrifugation with sucrose density gradient and mix with equal volume of saturated phenol: chloroform: isoamyl alcohol (25:24:1), and shake for 3~5 min in an ice bath.
2) Centrifuge at 8000 g for lOmin at 4℃, aspirate the upper aqueous phase, and then treat the same method for the second time, and then wash the upper aqueous phase with ether; remove the phenol for the second time, and then add 1/10◦volume of 2.5 mol/LKAc and 2 times the volume of ethanol to the aqueous phase, and then store it at -20℃.
2. Agarose gel preparation for electrophoretic purification method
1) Prepare a 1.2% agarose-TBE gel.
2) Add 40~80ul of electrophoresis sample solution to the RNA tube after centrifugation to remove ethanol, and apply the sample for electrophoresis. The electrophoresis solution was TBE at 60~100V for 3~6 hours.
3) Cut off the gel strip containing the viral RMA region, put it into a dialysis bag, add 0.5~2 ml of TBE into the bag, seal both ends of the bag with a thread or ordinary wooden or plastic clips, and put it into the electrophoresis tank for electrophoresis again. After about 1 hour, the RMA will come out of the gel, and the RNA in the bag will be aspirated, and KC1 will be added to 0.25 mol/L, and 2 times the volume of ethanol will be added, and then the bag will be stored in a cool place.
3. B encephalitis virus RNA purity check
1) The identification of B encephalitis virus RNA infectivity can be accomplished by titration in mouse brain, or by determining its mRNA activity in a wheat germ cell-free protein synthesis system.
2) To determine the molecular mass size, encephalitis B virus RNA is a single band on agarose gel electrophoresis. rRNA size markers can be used as 18SRNA and 28SRNA from normal animal cells, which have a molecular mass of 0. 7X106Da and 1. 65Xl06Da, respectively, in mouse cells.