7. Place tubes on a heat block at 80
C for 50 min.
8. Freeze at À20
C for 30 min.
9. Thaw and vortex the tubes for 3 min. Centrifuge for 5 min at
16,000 Â g.
10. Pipette 20μL of Qiagen Protease into the bottom of a 1.5 mL
DNA LoBind tube.
11. Add 200μL of the heat-killed, decontamination sputum from
step 9.
12. Add 200μL of Buffer AL and mix by pulse vortexing for 15 s.
13. Incubate at 56
C for 10 min. Briefly spin down tube to collect
sample.
14. Add 200μL of ethanol (96–100%) to the sample, mix by pulsevortexing for 15 s, and briefly centrifuge to collect.
15. Insert a QIAamp Mini spin column into a 2 mL collection tube
and carefully add the sample to the column without wetting the
rim. Close the cap and centrifuge at 6000 Â g for 1 min.
16. Remove QIAamp column to a fresh 2 mL collection tube and
discard the tube containing the flow-through (see Note 7).
17. Add 500μL of Buffer AW1 to the column, spin at 6000 Â g for
1 min, remove QIAamp column to a fresh 2 mL collection
tube, and discard the tube containing the flow-through (see
Note 7).
18. Add 500μL of Buffer AW2 to the column, spin at full speed for
1 min, remove QIAamp column to a fresh 2 mL collection
tube, and spin at top speed for 1 min; discard the tube containing the flow-through.
19. Place QIAamp column in a new DNA LoBind tube, add 100μL
of Buffer AE, incubate at room temperature for 5 min, and
then spin at 6000 Â g for 1 min.
20. Label lids of Qubit assay tubes, for the number of samples plus
2 standards.
21. Prepare Qubit working solution by diluting Qubit dsDNA HS
reagent 1:200 with Qubit dsDNA HS Buffer in a 50 mL plastic
tube and allow for 200μL/sample or standard.
22. Add 190μL of Qubit working solution to each standard tube.
23. Add 199μL of Qubit working solution to each sample tube.
24. Add 10μL of each standard to the appropriate tube and 1μL of
each sample.
25. Mix by vortexing for 2–3 min, take care not to create bubbles.
26. Briefly spin tubes down in a microfuge. Incubate at room
temperature for 2 min.
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Amanda Claire Brown
C for 50 min.
8. Freeze at À20
C for 30 min.
9. Thaw and vortex the tubes for 3 min. Centrifuge for 5 min at
16,000 Â g.
10. Pipette 20μL of Qiagen Protease into the bottom of a 1.5 mL
DNA LoBind tube.
11. Add 200μL of the heat-killed, decontamination sputum from
step 9.
12. Add 200μL of Buffer AL and mix by pulse vortexing for 15 s.
13. Incubate at 56
C for 10 min. Briefly spin down tube to collect
sample.
14. Add 200μL of ethanol (96–100%) to the sample, mix by pulsevortexing for 15 s, and briefly centrifuge to collect.
15. Insert a QIAamp Mini spin column into a 2 mL collection tube
and carefully add the sample to the column without wetting the
rim. Close the cap and centrifuge at 6000 Â g for 1 min.
16. Remove QIAamp column to a fresh 2 mL collection tube and
discard the tube containing the flow-through (see Note 7).
17. Add 500μL of Buffer AW1 to the column, spin at 6000 Â g for
1 min, remove QIAamp column to a fresh 2 mL collection
tube, and discard the tube containing the flow-through (see
Note 7).
18. Add 500μL of Buffer AW2 to the column, spin at full speed for
1 min, remove QIAamp column to a fresh 2 mL collection
tube, and spin at top speed for 1 min; discard the tube containing the flow-through.
19. Place QIAamp column in a new DNA LoBind tube, add 100μL
of Buffer AE, incubate at room temperature for 5 min, and
then spin at 6000 Â g for 1 min.
20. Label lids of Qubit assay tubes, for the number of samples plus
2 standards.
21. Prepare Qubit working solution by diluting Qubit dsDNA HS
reagent 1:200 with Qubit dsDNA HS Buffer in a 50 mL plastic
tube and allow for 200μL/sample or standard.
22. Add 190μL of Qubit working solution to each standard tube.
23. Add 199μL of Qubit working solution to each sample tube.
24. Add 10μL of each standard to the appropriate tube and 1μL of
each sample.
25. Mix by vortexing for 2–3 min, take care not to create bubbles.
26. Briefly spin tubes down in a microfuge. Incubate at room
temperature for 2 min.
466
Amanda Claire Brown