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    E-Z 96® Viral RNA Protocol with Centrifugation

    互联网

    1705

    实验原理

     

    The E-Z® Viral RNA Kits use reversible binding properties of HiBind® matrix, a new silica-based, time saving spin technology material. Combined with the speed of minicolumn spin technology or vacuum manifold, multiple samples can be processed at same time. The sample is lysed first under highly denaturing buffer conditions so that RNases will be inactivated, and the intact viral RNA is protected from degrading. After adjusting the buffer condition, the samples are loaded to the HiBind® RNA Plate. With a brief centrifugation or vacuum, the samples pass through the plate and the viral RNA binds to the Hibind® matrix. After two washing steps, purified viral RNA will be eluted with RNase-free water.

    实验试剂

     

    Materials supplied by user

    1. 96-100% ethanol

    2. β-Mercaptoethanol

    实验设备

     

    Materials supplied by user

    1. Multichannel pipet

    2. RNase-free filter pipette tips

    3. Reagent reservoirs for multichannel pipets

    4. Centrifuge with suitable rotor for 96-well plate.

    5. Disposable latex gloves

    6. RNase-Free 1.2 ml 96-well plate

    7. Adhesive sealing film for microplate

    8. 2ml 96-well deep well plate

    实验步骤

     

    1. Use a 1.2 ml 96-well plate to prepare sample. Add 150 ul plasma, cell free body fluid, cell culture or urine into each well of the 96-well plate.

    2. Pipet 10ul of reconstituted OB Protease solution into each well of the 96-well plate.

    3. Pipet 500 ul QVL lysis buffer into the each well of the 96-well plate. Keep the microplate flat on the bench, shake vigorously back and forth for 30 seconds. Rotate the plate 90 and shake the plate for another 30 secondsB.

    Note: Add 20 ul Buffer/ß-mercaptoethanol with each 1ml QVL buffer before use. Make sure that Carrier RNA is added to QVL Lysis buffer according to the instructions.

    4. Seal the plate with sealing film. Incubate at room temperature for 5-10 minutes.

    5. Spin at 500 x g briefly to collect any liquid.

    6. Add 350ul of absolute ethanol (96-100%) to the sample, mix throughly by vortexing for 30 seconds. Centrifuge briefly to collect any liquid droplets from lid.

    7. Carefully apply 500ul samples from step 6 (including any precipitate) to each well of the HiBind® RNA plate.

    8. Seal the HiBind® RNA plate with new sealing film. Load the HiBind® RNA plate with 2ml 96-well plate into the plate holder, and place the whole assembly into the rotor bucket of the centrifuge. Spin at 5500 x g for 5 minutes at room temperature.

    9. Remove the sealing film and repeat steps 7 and 8.

    10. Remove the sealing film. Wash plate with Wash Buffer I by pipetting 750 ul directly into the each well of the HiBind® RNA plate . Seal the plate with a new sealing film. Centrifuge at 5500 x g for 5 minutes at room temperature.

    11. Place HiBind® RNA plate on top of another clean 2ml 96-well plate (Not supplied). Remove the sealing film and add 500 ul Wash Buffer II diluted with ethanol to each well of HiBind® RNA plate. Seal the plate with a new sealing film. Centrifuge at 5500 x g for 5 minutes at room temperature.

    Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

    12. Remove the sealing film. Add another 500ul of RNA Wash Buffer II to each well of HiBind® RNA plate. Seal the plate with a new sealing film. Centrifuge at 5500 x g for 10 minutes at room temperature.

    Note: It is very important to dry the HiBind® RNA plate before the elution because the residual ethanol might interfere with downstream applications.

    13. Elution of RNA: Remove the sealing film and place the HiBind® RNA plate onto the microtube rack containing 1.2ml microtubes (supplied with kit).

    14. Add 50-70 ul of DEPC-treated water to each well, and seal the HiBind® RNA plate with new sealing film(supplied with kit). Make sure to add water directly onto RNA matrix. Incubate for 1 minute at room temperature. Centrifuge at 5500 x g for 5 minutes at room temperature to elute RNA.

    15. Remove the sealing film. Repeat step 13 and 14 for second elution.

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