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        Purification of Inhibitor-Free Soil DNA Obtained by other Methods

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        678

        实验步骤

         

        1. Dissolve the DNA pellet with 200 ul of Elution Buffer or Buffer TE. Important: DNA pellet should be dissolved in Elution Buffer or Buffer TE(pH8.0).

        2. Add 50 ul inhibitor Removal Resin to the sample and vortex to mix well.

        Note: Completely resuspend HTR Reagent by shaking the bottle before use.

        3. Incubate at room temperature for 2 min and centrifuge at maxi speed ($13,000 x g) for 2 min to remove Inhibitor Removal Resin.

        4. Carefully transfer the supernatant into a new tube.

        Note: If supernatant still have dark color from soil, perform the HTR treatment again by repeating step 2-4.

        5. Transfer cleared supernatant to a new 2 ml tube.

        6. Add equal volume of XP2 Buffer, mix by vortexing. For example: if the sample from step 12 is 250 μl, add 250 μl Buffer XP2.

        7. Apply the sample from step 13 to a HiBind® DNA Column assembled in a 2 ml collection tube (supplied). Centrifuge at 10,000 x g for 1 min at room temperature. Discard flow-through liquid and re-use collection tube.

        8. Place the column into the same 2 ml collection tube from previous step and add 300 μl XP2 Buffer. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and collection tube.

        9. Place column into a new 2 ml collection tube (supplied) and wash by adding 700 μl SPW Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000 x g for 1 minute. Discard flow-through liquid and re-use collection tube in next step.

        Note: SPW Wash Buffer is provided as a concentrate and must bediluted with absolute ethanol as indicated on the bottle and page 4.

        10. Repeat step 16 with a second 700 μl SPW Wash Buffer.

        11. Discard liquid and re-insert the column to the empty collection tube, centrifuge the column at full speed (13,000 x g) for 2 min at room temperature. This step is critical in removing traces of ethanol that will interfere with downstream applications.

        12. Place column into a clean 1.5 ml microcentrifuge tube (not supplied). Add 30-100 μl Elution Buffer directly onto the center of HiBind® matrix. Incubate at 65°C for 10-15 minutes.

        13. Centrifuge at full speed (13,000 x g) for 1 min to Elute DNA.

        14. Repeat elution step 12-13 with a second 30-100 μl Elution Buffer.

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