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E.Z.N.A.® Viral RNA Spin Protocol

互联网2013-11-13

1135

实验试剂

1. Absolute ethanol ( 96-100%)

实验设备

1. Sterile RNase-free pipette tips and microcentrifuge tubes

2. Table top microcentrifuge at room temperature.

3. 2-mercaptoethanol

4. Disposable latex gloves

5. (Optional)Vacuum manifold with standard leur adaptor

实验步骤

1. Add 560 ul QVL Lysis buffer, 5.6 ul Carrier RNA and 10 ul 2-mercaptoethanol into a 1.5 ml micro-centrifuge tube.

Note: QVL Lysis Buffer, Carrier RNA and 2-mercaptoethanol can be premixed together. The premixed lysis buffer can stored at 2-8°C for 1 week. Increase the amount of Lysis Buffer proportionally if the sample volume is larger than 140 ul.

2. Pipet 140 ul plasma, cell free body fluid or urine into the micro-centrifuge tube containing Lysis Buffer. Mix throughly by vortexing at maxi speed for 30 seconds.

3. Incubate at room temperature for 5-10 minutes. Briefly spin to collect any liquid from lid.

4. Add 560 ul of absolute ethanol (room temperature, 96-100%) to the sample, mix throughly by vortexing at maxi speed for 30 seconds.

5. Apply the 650 ul of the mixture (including any precipitate) to a HiBind® RNA column assembled in a 2 ml collection tube (supplied). The maximum capacity of the HiBind® RNA column is 800 ul. During the procedure, work carefully but quickly. Centrifuge at 10,000 x g for 30 seconds. Discard flow-through.

6. Repeat step 5 until all the lysate has been loaded into the column and passed through the column.

7. Place the column into a new 2 ml collection tube (from step 6), and add 500 ul RWA Wash Buffer diluted with ethanol. Centrifuge as above and discard flowthrough.

Note: RWA Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle or before starting on page 4 for directions.

8. Place the column into a new 2 ml collection tube and add 500ul RWB Wash Buffer diluted with ethanol. Centrifuge as above and discard the flow-through.

Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle or before starting on page 4 for directions.

9. Place the column back into the collection tube, centrifuge the empty column at full speed(no more than 12,000 x g ) for 3 min to completely dry the HiBind. matrix.

10. Transfer the column to a clean 1.5 ml microfuge tube (not supplied) and add 30-50 ul DEPC water (supplied with kit) directly onto column matrix. Allow the column to incubate for 3 to 5 min at room temperature. Centrifuge at 10,000 x g for 1 min to elute RNA. Store Purified RNA at -70°C.

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