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        Isolate DNA from NRBC Blood from Buccal Swab or dried blood spots collected with filter paper

        互联网

        668

        实验试剂

         

        1. Absolute Ethanol - approximately 0.3 ml per sample

        2. RNase A(Optional) - Prepare a stock of RNase A at 50mg/ml

        实验设备

         

        1. Tabletop microcentrifuge and sterile 1.5 ml tubes

        2. Water bath - set to 65°C

        实验步骤

         

        1. Place the buccal swab or paper contains dried blood spot in a 2 ml centrifuge tube and add 500 ul RS1 Buffer in the tube. If RNA-free DNA is required for downstream application, add 4 ul RNase A (25mg/ml) into the sample.

        2. Add 25ul OB Protease and 500ul Buffer BL into sample. Mix by vortexing at maxi speed for 30s.

        3. Incubate at 65°C for 10 minutes. Collect any liquid drop from lid by brief centrifugation.

        4. Centrifuge at 14,000 x g for 10 minutes. Carefully transfer the supernatant to a new 1.5 ml tube.

        5. Add equal volume of absolute ethanol (room temperature, 96-100%) to the sample and mix by vortexing at maxi speed for 15 seconds. Collect any liquid drop from lid by brief centrifugation.

        6. Assemble an HiBind DNA column in a 2 ml collection tube (provided). Add 100ul of Equitation Buffer to each column. Wait 3-4 minutes at room temperature. Centrifuge at maximum speed for 30 seconds.

        7. Carefully apply entire sample into HiBind® DNA. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and collection tube.

        8. Place the column into a new 2 ml collection tube (provided). Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and collection tube.

        9. Place the column into a new 2 ml collection tube (provided). Add 500ul of HB Buffer into the column. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and reuse the collection tube in next step.

        10. Place the column into a same 2 ml collection tube from step 7. Add 700ul of DNA Wash Buffer diluted with absolute ethanol into the column. Centrifuge at 8000 x g for 1 minute. Discard the flow-through and reuse the collection tube in next step.

        Note: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle and page 3. If refrigerated, the diluted wash buffer must be brought to room temperature before use.

        11. Place the column into a same 2 ml collection tube from step 8. Centrifuge at maximum for 2 minutes to completely dry the HiBind® DNA column. Discard the flow-through and the collection tube.

        12. Place the column into a sterile 1.5 ml microcentrifuge tube and add 50-100 ul of preheated 65°C) Elution Buffer (10mM Tris-HCl, pH 8.5). Allow tubes to sit for 5 min at room temperature.

        13. To elute DNA from the column, centrifuge at maximum for 1 min. Retain flowthrough containing the DNA. Place column into a second 1.5 ml tube. Elute DNA again as step 10-11. Discard column and store the eluted DNA at -20°C.

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