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Blood & Body Fluid DNA Protocol

互联网2013-11-13

820

实验试剂

Materials provided by user

1. Tabletop microcentrifuge

2. Sterile 1.5ml centrifuge tubes

4. RNase stock solution

5. Absolute ethanol

6. OB Protease stock solution

实验步骤

1. Transfer the sample into a sterile microcentrifuge tube and bring the volume up to 250 μl with 10mM Tris-HCl, PBS, or Elution Buffer (provided).

2. Add 25μl of Reconstituted OB Protease and 250 μl of Buffer BL. Vortex at maximum speed for 15 seconds to mix thoroughly. If RNA-Free Genomic DNA is required, add 5μl of RNase A (50mg/ml) to each sample.

3. Incubate the sample at 65℃ for 10 minutes. BRIEFLY vortex the tube once during incubation.

Note: During incubation step 5 can be processed.

4. Add 260 μl of absolute ethanol (RT, 96-100%) to lysate. Vortex at maximum speed for 20 seconds to mix thoroughly. Briefly centrifuge the tube to collect any drops from the inside of the lid.

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

6. Transfer the lysate from step 4 into the column, and centrifuge at 10,000 x g for 1 min to bind DNA. Discard the collection tube and flow-through liquid.

7. Place the HiBind^® DNA Mini Column into a NEW provided 2 ml collection tube. Add 500μl of Buffer HB, and centrifuge as above. Discard collection tube, and flowthrough liquid.

8. 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 at 10,000 x g for 1 min. Discard collection tube, and flow-through liquid.

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

9. Using a NEW 2 ml collection tube, REPEAT step 8 with an ADDITIONAL 700μl of DNA Wash Buffer diluted with absolute ethanol, and centrifuge as above. Discard flow through. (Collection tube will be re-used in the following step)

10 Place the empty column into the SAME 2ml collection tube from step 9, centrifuge at a maximum speed of (13,000 x g) for 2 min to dry the column.

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

11. Place the HiBind^® DNA Mini Column into a sterile 1.5 ml microfuge tube, and add 100-200μl of preheated (65℃) Elution Buffer (10mM Tris-HCl, pH 8.5, 65℃). Allow to sit at room temperature for 5 minutes.

12. To elute DNA from the HiBind^® DNA Mini Column, centrifuge at (13,000 x g) for 1 min. Retain flow-through containing the DNA. Place the column into a new 1.5ml tube. Elute DNA again as indicated in previous step. Discard column, and store the Eluted DNA at -20℃.

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