Rapid isolation of total RNA from mammalian cells
SDS lyses the cells and extracts the total RNA by acid phenol graded extraction, which is a suitable method for treating cells with relatively low mRNA content and high endogenous RNase activity, with minimal loss of RNA, and is simple and easy to use, and can be used for treating multiple samples at the same time. For most cell lines, the RNA yield is 100~200ug in a 90mm diameter dish.
Operation method
Rapid isolation of total RNA from mammalian cells
Materials and Instruments
Eucalyptus Magnesium Ion Phosphate Buffer Salt Solution EDTA SDS Sodium Acetate Water Balance Phenol Ethanol Tris-Cl NaCL Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
1) Eucalyptus magnesium ionized phosphate buffer salt solution (PBS)
2)10 mmol/L EDTA (pH8.0 )
3)0.5% SDS
4)0. l mol/L sodium acetate (pH 5.2 )
5)Phenol for water balance
6)ethanol
7)l mol/L Tris-Cl (pH8.0 )
8)5 mol/L NaCL
II Methods of operation
1) Aspirate the culture medium and rinse the monolayer of cells in the cell culture dish with 7 ml of ice pre-cooled calcium and magnesium ion free phosphate buffered saline solution (PBS), and repeat the rinsing once. The plate should be kept on ice until all monolayers have been rinsed.
2) Add 2 ml of 10 mmol/L EDTA (pH 8.0) and 0.5% SDS to each 90 mm diameter petri dish, and scrape the lysate into a disposable 15 ml centrifuge tube with a scraper.
3 ) Rinse the plate with 2 ml of 0.1 mol/L sodium acetate (pH 5.2), 10 mmol/L EDTA (pH 8.0) and transfer the rinse solution to the centrifuge tube containing cell lysates as described above.
4 ) Shake 4 ml of water-balanced phenol for 2 mm at room temperature to mix with the cell lysate, and the cellular DNA will form a white filamentous precipitate.
5 ) Centrifuge the lysate at 5000r/mm for 10mm at 4℃ to separate the phases, and a dense DNA layer can be seen between the two phases.
6 ) Transfer the upper layer (aqueous phase) with a Pasteur pipette to another centrifuge tube containing 440u1 of ice-cooled lmol/L Tris-Cl (pH 8.0) and 180ul of 5mol/L NaCl.
7 ) Add 2 times the volume of ice pre-cooled ethanol, mix well and leave at 0°C for at least 30 min.
8 ) Centrifuge at 5000r/mim for l0min at 4°C to precipitate the RNA, discard the ethanol, and invert the tube upright until all the ethanol has evaporated.
9 ) Resolve RNA with 200ul of ice-cooled TE (pH 8.0), transfer to a sterilized microcentrifuge tube and add 5mol/L NaCl, 50ul of ice-cooled ethanol.
1 0 ) centrifuged in a microcentrifuge at 12000 feet for 5 min at 4°C to collect the RNA, the
11) Aspirate out all the ethanol and place the uncapped centrifuge tube on the lab bench for a few minutes to allow the last remaining traces of ethanol to evaporate.
1 2 ) Resolve the RNA with the desired buffer.