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Stool DNA Protocol (for pathogen detection)

互联网2013-11-13

812

实验试剂

1. Equilibrate sterile dH₂ O or 10 mM Tris pH 8.5 at 65°C.

2. Absolute (96%-100%) ethanol

3. RNase A stock solution at 20 mg/mL

4. Isopropanol (100%)

实验设备

1. Microcentrifuge capable of at least 13,000 x g

2. Nuclease-free 1.5 mL or 2 mL microfuge tubes

3. Water bath or heating block preset to 65°C and 70°C

实验步骤

1. Weigh up to 50-100 mg of stool sample in a 2 mL centrifuge tube containing 200 mg of glass beads and place the tube on ice.

Note: If the sample is liquid, pipet 200ul of sample into the centrifuge tube. Cut the end of the pipet tip to make pipetting easier. If the sample is frozen, use a spatula to scrape the sample into the tube. Do not thaw the frozen sample until the SP1 Buffer/Proteinase K is added into the tube.

2. Add 300 ul Buffer SP1 followed by then adding 10 ul of Proteinase K solution. Vortex at maxi speed for 5 minute or until the stool sample is throughly homogenized.

3. Incubate at 70°C for 10 min (13 min if frozen). Mix sample twice during incubation by vortexing the tube. Optional: For isolation of DNA from gram positive bacteria, do a second incubation at 90°C for 5 minutes.

4. Incubate the sample on ice for 2 minutes.

5. Add 100 μl of Buffer SP2. Mix the sample throughly by voretxing the tube for 30 seconds.

6. Incubate the sample on ice for 5 minutes.

7. Centrifuge at full speed (13,000-20,000 x g) in a microcentrifuge for 5 minute to pellet the stool particles.

8. Carefully aspirate supernatant to a new 1.5 mL microfuge tube (not supplied), making sure not to disturb the pellet or transfer any debris.

9. Add 200 ul of HTR Reagent. Mix the sample by vortexing the tube for 10 seconds.

Important: HTR reagent must be throughly suspended before being dispense from bottle. Tip: Use 1ml pipettor and cut off the end of 1ml tip to make it easier for pipetting the HTR reagent.

10. Incubate at room temperature for 2 minutes.

11. Centrifuge at full speed (>13,000 x g) for 2 minutes to pellet the inhibitors absorb to HTR Reagent.

12. Transfer 250 ul supernatant to a new 2.0 ml tube.

13. Optional: If RNA-free DNA is required, add 10 ul RNase A and mix thoroughly by vortexing. Incubate at 70°C for 3 minutes.

14. Add 250 ul of BL Buffer followed by 250μl of absolute ethanol to the lysate. Miix sample throughly by vortexing the tube for 10 seconds.

15. Apply entire sample from step 14 including any precipitation that may have formed, to a HiBind® DNA column assembled in a 2 mL collecting tube (supplied). Centrifuge at full speed (>13,000 x g) for 1 min at room temperature. Discard flow-through liquid and collection tube.

16. Place column into a new 2 mL collection tube (supplied) and wash the column by adding 500 ul HB Buffer. Centrifuge at full speed (>13,000 x g) for 1 min. Discard flow-through liquid and collecting tube in next step.

17. Place the HiBind® DNA column into a new collection tube (supplied). Add 750μl of DNA Wash Buffer into the column and centrifuge at full speed (>13,000 x g) for 1 min. Discard liquid and re-insert the column to the empty collection tube.

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 DNA Wash Buffer must be brought to room temperature before use.

18. Centrifuge the column at full speed (>13,000 x g) for 2 min at room temperature to dry the column.

This step is critical in removing traces of ethanol that will interfere with downstream applications.

19. Place column into a clean 1.5 mL microfuge tube (not suplied). To elute DNA add 200 ul of Elution Buffer (10 mM Tris buffer, pH 8.5) preheated to 60°C -70°C directly onto the HiBind® matrix. Allow to soak for 2 min at room temperature. Centrifuge at full spped ($13,000 x g) for 1 min to Elute DNA.

Tip: for maximum PCR robustness, it is recommended to add BSA to a final concentration of 1ug/ul for the PCR reaction mixture. Hot-start PCR is also recommended to increase the specificity. Try to use minimal amount of elute possible for downstream applications.

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