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        RNA Purification Protocol for 1-2 x 107 cultured cells

        互联网

        971

        实验原理

         

        E.Z.N.A.® SQ Tissue RNA uses a highly efficient solution based system to provide a convenient, fast, reliable and non-toxic method to isolate high quality RNA from various samples. Samples are first lysed with RCL buffer. Cellular proteins and genomic DNA are removed by precipitation with PNP Buffer while RNA will remain in solution. RNA is further purified by isopropanol precipitation.

        实验试剂

         

        1. Isopropanol

        2. 70% ethanol

        实验设备

         

        1. Microcentrifuge capable of 3,000 x g

        2. 15 ml centrifuge tube

        3. Ice bath

        4. Ground pestle

        实验步骤

         

        1. Remove the media and collect cells.

        For cells grown in suspension, pellet the cell by centrifugation and discard the media, add 3 ml RCL. Pipetting up and down 3-5 times to lyse the cell.

        Note: Leave 0.1-0.2 ml media or salt balance buffer to fully resuspend the cell pellet before addition of RCL Buffer.

        For cells grown in monolayer, cell can be directly lysed by directly adding the 3 ml of RCL Buffer directly into the culture plate or flask. lyse and homogenize the sample by shaking or vortexing. Transfer the cell lysate into a 15 ml centrifuge tube

        2. Add 1 ml of PNP Buffer to the cell lysate. Mix the sample gently by inverting the tube 10 times.

        3. Place the tube on ice for 5 minutes.

        4. Centrifuge at max speed 3,000-5,000 x g for 15 minutes at room temperature. The precipitated protein and DNA will form a tight pellet.

        5. Transfer the supernatant to a new 15 ml high speed centrifuge tube that containing 3 ml of 100% isopropanol.

        6. Gently mix the solution by inverting the tube 30-40 times.

        7. Centrifuge at 3,000-5,000 x g for 15 at room temperature.

        8. Pour of the supernatant and drain the tube briefly on a clean absorbent paper. Add 3 ml of 70% ethanol and invert the tube few times to wash the RNA pellet.

        9. Centrifuge at 3,000-5,000 x g for 10 minutes at room temperature. Carefully pour off the ethanol. Pellet may be very loose at this point so pour slowly and watch the pellet.

        10. Invert the tube on a clean adsorbent paper and air dry the pellet for 10-15 minutes.

        11. Add 250 ul of DEPC Water and vortex for 1 minutes to mix.

        12. Incubate sample on ice for at least 30 minutes. Vortex sample vigorously for 10 seconds and pulse spin.

        13. Store RNA at -70°C.

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