Isolation of RNA based on size
Isolation of RNA based on size
RNA samples may be denatured by treatment with formamide and by electrophoretic separation on gels containing formaldehyde, a method modified from Lehrachetal. (1977), Goldberg (1980), Seed (1982a), and Rosen et al, (1990).RNA was separated electrophoretically on gels containing 2.2 mol/L formaldehyde. RNA was separated by electrophoresis on a gel containing 2.2 mol/L formaldehyde. RNA was separated by electrophoresis on a gel containing 2.2 mol/L formaldehyde.
Operation method
Isolation of RNA by size: electrophoresis of RNA on agarose gels containing formaldehyde
Principle
RNA samples may be denatured by treatment with formamide and by electrophoretic separation on gels containing formaldehyde, a method modified from Lehrachetal. (1977), Goldberg (1980), Seed (1982a), and Rosen et al, (1990).RNA was separated electrophoretically on gels containing 2.2 mol/L formaldehyde. RNA was separated by electrophoresis on a gel containing 2.2 mol/L formaldehyde.
Materials and Instruments
RNA Sample RNA Size Standard Reference Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethidium Bromide Formaldehyde Formamide Formaldehyde Gel Electrophoresis Spiking Buffer MOPS Electrophoresis Buffer
Agar Enamel Gel Horizontal Electrophoresis Apparatus Transparent Ruler Water Bath
1. Buffers and solutions
Ethidium bromide (200 μg/ml)
Formaldehyde
Formamide
10X Formaldehyde Gel Electrophoresis Spiking Buffer
10X MOPS Electrophoresis Buffer
2. Gel
Agar enamel gel containing 2.2 mol/L formaldehyde
3. nucleic acids and oligonucleotides
RNA samples
RNA Size Reference
4. Specialized equipment
Horizontal electrophoresis apparatus
Transparent ruler
55°C water bath
II. METHODS
1. Establish the denaturation reaction in a sterilized microcentrifuge tube.
RNA (up to 20 μg) 2.0 μl
10X MOPS electrophoresis buffer 2.0 μl
Formaldehyde 4.0 μl
Formamide 10.0 μl
Ethidium bromide (200 μg/ml) 1.0 μl
2. Cap the microcentrifuge tube tightly, warm the RNA liquid at 55℃ for 60 min, cool the sample in ice water for 10 min, then centrifuge for 5s and settle all the liquid at the bottom of the microcentrifuge tube.
3. Add 2 μl of 10X Formaldehyde Gel Spiking Buffer and place the tube back on ice.
4. Load the agarose/formaldehyde gel into a horizontal electrophoresis tank and add enough 1X MOPS Electrophoresis Buffer to cover the gel by about 1 mm. electrophoresis for 5 min (5 V/cm). Add RNA samples to the gel spiking wells, leaving the two outermost lanes on either side of the empty gel, and spiking the RNA standard size reference.
5. Immerse the gel in 1X MOPS electrophoresis buffer and electrophoresis at 4~5 V/cm until the bromophenol blue is removed by about 8 cm (4~5 h). Higher voltages during electrophoresis will result in blurred bands. Because the pH value of the electrophoresis buffer changes during electrophoresis, the electrophoresis tank is loaded and the buffer is continuously moved from one chamber to another by a peristaltic pump. The buffer is changed every hour.
6. Place the gel on a piece of Saran Membrane Observe the RNA on a UV transilluminator Compare the stained gel with the UV transilluminated photographs using a clear straightedge.
7. Photograph and measure the distance from each RNA band on the photograph to the sample well. Plot the fitness distances of the RNA bands against the logarithm of the RNA fragment size, and use the resulting curves to calculate the size of the RNA molecules by hybridization after transferring them from the gel to the solid-phase support.
8. Transfer the solidified RNA to the solid-phase support via an upper or lower capillary.