| Laboratory of P.J. Hansen,Dept. of Animal Sciences, University of Florida
RNA_extraction.htm">http://www.animal.ufl.edu/hansen/Protocols/RNA_extraction.htm
Reagents and Equipments
TRIzol Reagent (Life Technologies cat# 15596-026) or TRI reagent (Sigma cat # T-9424)
DEPC (RNase free) water or 0.5% SDS solution in DEPC treated water
Chloroform (Fisher )
Isopropyl alcohol (2-Propanol) (Fisher)
75% Ethanol (in DEPC treated water)
Sterile or RNase treated pipette tips, microcentrifuge tubes, and pestles or motorized homogenizer.
Microcentrifuge
1. Homogenization for Cell Suspensions
a. Place 1 ml aliquots of the cell suspension in sterile RNase free 1.5 ml microcentrifuge tubes.
b. Centrifuge for 1 minute to pellet the cells.
c. Pour off the supernatant.
d. Add 1 ml of TRIzol or TRI reagent to the tubes.
e. Lyse cells by repetitive pipetting.
f. Centrifuge homogenate at 12000 x g for 10 minutes at 4 oC.
g. Transfer the homogenate in a sterile microcentrifuge tube.
h. If this RNA will be used for RT-PCR, repeat steps f and g twice.
Modification for tissues
a. Add 1 ml of TRIzol or TRI reagent to every 50-100 mg of tissue. Sample volume should not exceed 100 μl. If you using 1.5 -2 ml microcentrifuge tube and pestle for homogenization, start with 500 μl of TRIzol or TRI reagent, then add remaining 500 μl.
b. Homogenize the samples using a sterile or RNAse free plastic, glass pestles or power homogenizer and tubes.
Modification for monolayers
a. Lyse cells directly in a culture dish by adding 1 ml of TRIzol or TRI reagent to a 3.5 cm diameter dish.
b. Pass the cells through a pipette several times.
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