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        E.Z.N.A.TM Fast Protocol For FFPE Tissue

        互联网

        1036

        实验原理

         

        E.Z.N.A.TM FFPE RNA Kit uses the reversible binding properties of HiBind® matrix, a new silica-based material, combined with the speed of mini-column spin technology. A specifically formulated buffer system allows RNA less than 20 bp to bind to the matrix. Samples are first lysed under denaturing conditions and then applied to the HiBind® spin columns to which RNA binds, while cellular debris, hemoglobin, and other proteins are effectively washed away. High quality RNA is finally eluted in sterile deionized water or low salt buffer.

        实验步骤

         

        1. Using a scalpel, trim excess paraffin off the sample block. Cut sections 5-10um thick. If the sample surface has been exposed to air, discard the first 2-3 sections.

        2. Immediately transfer 3-8 sections in a 1.5 or 2 ml tube. Add 250 ul Buffer FTL to the tissue, mix by vortexing.

        3. Incubate at 80°C for 15 min. Incubate for 1 minutes at room temperature.

        4. Immediately add 20 ul OB Protease to the tissue, mix by vortexing.

        5. Incubate at 55°C for 15-60 min.

        6. Centrifuge at 10,000 x g at room temperature for 3 minutes. The paraffin will form a thin layer on top of the lysate solution.

        7. Use a 1 ml pipette tip or large orifice tip to penetrate the paraffin layer, transfer 200 μl of lysate into a new 1.5 ml tube.

        8. Add 220 ul Buffer GTC and vortex to mix.

        9. Add 660 ul absolute ethanol and mix thoroughly by vortexing.

        10. Assemble an HiBind® RNA MicroElute column in a 2 ml collection tube (provided). Transfer 700ul of the sample from step 10 into the column including any precipitate that may have formed. Centrifuge at 10,000 x g for 1 min to bind RNA. Discard flow-through liquid and reuse the collection tube in step 12.

        11. Repeat step 11 until the entire sample has passed through the HiBind RNA MicroElute Column. Discard flow-through liquid and the collection tube.

        12. Place the column into a new 2 ml collection tube and wash by pipetting 500 ul of RWB Wash Buffer diluted with ethanol. Centrifuge at 10,000 x g for 1 min. Discard flow-through.

        Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, RWB Wash Buffer must be brought to room temperature before use.

        13. Using the same 2 ml collection tube, wash the column with a second 500 ul of RWB Wash Buffer. Centrifuge at maximum speed (10,000 x g) for 2 min to dry the column. This step is crucial for ensuring optimal elution in the following step.

        14. Place the column into a sterile 1.5 ml microcentrifuge tube and add 15-50 ul of DEPC Tteated Water. Allow tubes to sit for 3 min at room temperature.

        15. To elute RNA from the column, centrifuge at 10,000 x g for 1 min.

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