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E.Z.N.A.® Dried Blood DNA Spin Protocol

互联网2013-11-13

914

实验步骤

1. Cut or punch out the blood spot from the filter paper (up to 200ul of blood can be used per spot). Tear or cut the filter paper into small pieces and place them into a microfuge tube.

2. Add 250ul of PBS Buffer, and incubate at 65℃ for 1 hour, while vortexing every 20 min for proper mixing.

3. Add 25μl of OB Protease stock solution and mix well. Incubate for 30 min at 65℃ with occasional mixing.

Note: Step 6 can be performed during incubation time.

4. Centrifuge at 13,000 x g for 5 min at room temperature.

5. Transfer the supernatant to a clean microfuge tube and add ONE volume of Buffer BL followed by ONE volume of absolute ethanol. Vortex thoroughly to mix. Collect any drops remaining on the lid by briefly centrifuging.

6. Insert a HiBind^® DNA Mini Column into a 2 ml collection tube (provided). Add 100ul Equilibration Buffer into the column. Let the column sit for 4 minutes at room temperature. Spin at maximum speed for 20 seconds.

7. Transfer the lysate from step 5 into the column, and centrifuge at 10,000 x g for 1 min to bind the DNA. Discard the flow-through liquid and the collection tube.

8. Place the HiBind^® DNA Mini Column into a NEW 2 ml collection tube. Add 500:l of Buffer HB to the column, and centrifuge as above. Discard the flow-through and reuse the collection tube in the following step.

9. Place the HiBind^® DNA Mini Column into the SAME 2 ml collection tube from step 7, and wash by pipetting in 700 μl of DNA Wash Buffer diluted with ethanol. Centrifuge as above. Dispose of the collection tube and flow-through liquid.

NOTE: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle, and on page 4. If refrigerated, the diluted DNA Wash Buffer must be brought to room temperature before use.

10 Using a NEW collection tube (provided), wash the column with a second 700 μl of DNA Wash Buffer diluted with ethanol. Centrifuge as above. Discard flow-through liquid, and reuse the collection tube in the following step.

11. Place the empty column into the same 2 ml collection tube from step 10. Centrifuge at maximum speed (13,000 x g) for 2 minutes to completely dry the column. Discard the flow-through and the collection tube.

NOTE: This step is crucial for ensuring optimal elution in the following steps.

12 Place the column into a sterile 1.5 ml microcentrifuge tube, and add 100-200ul of preheated Elution Buffer (10mM Tris-HCl, pH 8.5, 65℃). Allow tubes to sit for 5 min at room temperature.

13. To elute DNA from the column, centrifuge at maximum speed (15,000 x g) for 1 min. Retain flow through containing the DNA. Discard column, and store the Eluted DNA at -20℃.

NOTE : Blood Spots from finger pricks usually contain no more than 50ul of blood, and yield approximately 500 ng to 1ug of DNA. This is usually sufficient for PCR analysis. To obtain higher DNA concentrations, elute with 50ul of preheated elution buffer (volumes lower than 50ul greatly reduce yields). Alternatively, the first eluate can be used to perform a second elution.

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