When working with many RNA samples or working with small amounts of total mammalian RNA (<50 μg), a batch chromatography method with oligo (dT)-cellulose is an option. This method uses a good grade of oligo (dT)-cellulose that is bound and washed at optimal temperatures. Many batch chromatography methods for purifying poly (A)+ RNA have been published over the years. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" Translated by Huang Peitang et al.

Operation method

Screening poly (A)+ RNA by batch chromatographic methods

Principle

When working with many RNA samples or working with small amounts of total mammalian RNA (<50 μg), a batch chromatography method with oligo (dT)-cellulose is an option. This method uses a good grade of oligo (dT)-cellulose that is bound and washed at optimal temperatures. Many batch chromatography methods for purifying poly (A)+RNA have been published over the years.

Materials and Instruments

Total RNA
Adsorption Wash buffer Ammonium acetate Ethanol Ice pre-cooled water NaCl TES
Colorimetric cups Microcentrifuge Oligo (dT)18-30-Cellulose Spinning wheel Water baths

Move

I. Materials

1. Buffers and solutions

Adsorption/washing buffer (TES buffer with 0.5 mol/L NaCl)

Ammonium acetate (10 mol/L)

Ethanol

Ice pre-cooled water

NaCl (5 mol/L)

TES

2. Nucleic acids and oligonucleotides

Total RNA

3. Specialized equipment

Colorimetric cup for measuring absorbance at 260 nm

Microcentrifuge with speed control

Oligo (dT _{)18-30-Cellulose}

Rotating Wheel

Water baths at 55°C and 65°C

ii. Methods

1. In a series of sterilized microcentrifuge tubes, adjust the volume of each sample of total RNA (upper limit 1 mg) to 600 μl with TES, seal the tubes, heat at 65℃ for 10 min, and then quickly cool to 0℃ on ice. Add 75 μl (0.1x volume) of 5 mol/L NaCl to each sample.

2. Add 50 mg (500 μl) of equilibrated oligo (dT)-cellulose to each tube, cap the tubes, and incubate for 15 min at room temperature on a rotating wheel.

3. Centrifuge the tubes in a microcentrifuge at 600-800 g (approx. 1500-2500 r/min) for 2 min at room temperature.

4. Transfer the supernatant to a series of clean centrifuge tubes and store on ice.

5. Add 1 ml of ice pre-cooled adsorption/washing buffer to the oligo (dT) precipitate. Disperse the precipitate by gentle vortexing. Place the capped tubes on a spinning wheel and incubate for 2 min at room temperature.

6. Centrifuge in a microcentrifuge at 600-800 g (approx. 1500-2500 r/min) for 2 min at room temperature and discard the supernatant. Repeat steps 5 and 6 twice.

7. Add 0.4 ml of ice-cooled, double-distilled sterilized water to the oligo(dT)-containing precipitate and resuspend the precipitate by gentle vortexing. Immediately centrifuge in a microcentrifuge at 4°C for 2 min.

8. Carefully aspirate the supernatant.

9. Recover bound poly(A ⁾⁺ RNA: Resuspend the precipitate with 400 μl of double-vaporized water, incubate at 55°C for 5 min, and then centrifuge at 4°C for 2 min.

10. Transfer the supernatant to a series of clean centrifuge tubes and repeat step 9 twice, combining all supernatants.

11. Add 0.2 times the volume of 10 mol/L ammonium acetate and 2.5 times the volume of ethanol to the supernatant and store at -20°C for 30 min.

12. Recover the precipitated poly(A ⁾⁺ RNA by centrifugation at 4°C, maximum speed, for 15 min. Carefully discard the supernatant, wash the precipitate with 70% ethanol, centrifuge briefly, aspirate the supernatant, uncap the tubes, and invert the tubes for a few minutes to allow most of the residual ethanol to evaporate.

13. Solubilize the RNA with a small volume of sterilized DEPC-treated water.

14. Estimate RNA concentration.

15. Save the sample.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/