20. Centrifuge at 12,000 Â g for 2 min to remove the ethanol and
air-dry the pellet at room temperature.
21. Dissolve the pellets from each tube in 200 μL RNase-DNasefree water and measure the DNA concentration using a NanoDrop spectrophotometer.
22. Digest up to 0.5 μg of the extracted DNA with 10 U/μL
BamHI-HF for up to 3 h at 37
C.
23. Run the digested DNA on 0.8% (w/v) agarose gel at 100 V for
35–45 min and visualize using a UV camera to assess the
quality of the extracted DNA (see Note 15).
24. Use the extracted genomic DNA to calculate the volume (μL)
required for the first standard sample which represents 10
8 copies/μL.
3.4 Genomic
Extractions from M.
tuberculosis and NTM
Using CTAB Extraction
Method
1. Culture 10 mL M. tuberculosis or NTM to late log phase in
7H9 supplemented with 10% ADC and 0.05% Tween-80 at
37
C, 180 rpm (see Notes 16 and 17).
2. Centrifuge culture in a 30 mL Universal tube at 2500 Â g for
20 min at RT and discard the supernatant.
3. Resuspend the pellet in 500 μL TE buffer and transfer to a
1.5 mL O-ring screw-capped tube. Alternatively, biomass from
a Middlebrook 7H11 agar plate can be removed with a sterile
loop and added to 500 μL TE buffer (see Notes 18 and 19).
4. Close tightly, decontaminate tube, seal the neck of the tube in
Parafilm and submerge in a 99
C water bath for 2 h to heat-kill
bacilli.
5. Freeze the pellet overnight at À80
C (see Note 20).
6. Thaw sample at room temperature and add 50 μL 10 mg/mL
lysozyme.
7. Mix gently by stirring with a pipette tip; pipetting or vortexing
will shear genomic DNA.
8. Incubate for 24 h in a 37
C water bath (see Note 21).
9. Pre-warm the CTAB/NaCl buffer to 65
C in a water bath.
Keep the water bath on at 65
C as will be needed for sample
incubation.
10. Add 15 μL RNase A to the sample, and mix by stirring with the
pipette tip, and incubate at 37
C for 30 min.
11. Add 70 μL 10% SDS and 5 μL 20 mg/mL proteinase K to the
sample. Mix gently by stirring the pipette tip.
12. Incubate for 15 min in the 65
C water bath and centrifuge
briefly.
DNA Isolation from Mycobacteria
69
air-dry the pellet at room temperature.
21. Dissolve the pellets from each tube in 200 μL RNase-DNasefree water and measure the DNA concentration using a NanoDrop spectrophotometer.
22. Digest up to 0.5 μg of the extracted DNA with 10 U/μL
BamHI-HF for up to 3 h at 37
C.
23. Run the digested DNA on 0.8% (w/v) agarose gel at 100 V for
35–45 min and visualize using a UV camera to assess the
quality of the extracted DNA (see Note 15).
24. Use the extracted genomic DNA to calculate the volume (μL)
required for the first standard sample which represents 10
8 copies/μL.
3.4 Genomic
Extractions from M.
tuberculosis and NTM
Using CTAB Extraction
Method
1. Culture 10 mL M. tuberculosis or NTM to late log phase in
7H9 supplemented with 10% ADC and 0.05% Tween-80 at
37
C, 180 rpm (see Notes 16 and 17).
2. Centrifuge culture in a 30 mL Universal tube at 2500 Â g for
20 min at RT and discard the supernatant.
3. Resuspend the pellet in 500 μL TE buffer and transfer to a
1.5 mL O-ring screw-capped tube. Alternatively, biomass from
a Middlebrook 7H11 agar plate can be removed with a sterile
loop and added to 500 μL TE buffer (see Notes 18 and 19).
4. Close tightly, decontaminate tube, seal the neck of the tube in
Parafilm and submerge in a 99
C water bath for 2 h to heat-kill
bacilli.
5. Freeze the pellet overnight at À80
C (see Note 20).
6. Thaw sample at room temperature and add 50 μL 10 mg/mL
lysozyme.
7. Mix gently by stirring with a pipette tip; pipetting or vortexing
will shear genomic DNA.
8. Incubate for 24 h in a 37
C water bath (see Note 21).
9. Pre-warm the CTAB/NaCl buffer to 65
C in a water bath.
Keep the water bath on at 65
C as will be needed for sample
incubation.
10. Add 15 μL RNase A to the sample, and mix by stirring with the
pipette tip, and incubate at 37
C for 30 min.
11. Add 70 μL 10% SDS and 5 μL 20 mg/mL proteinase K to the
sample. Mix gently by stirring the pipette tip.
12. Incubate for 15 min in the 65
C water bath and centrifuge
briefly.
DNA Isolation from Mycobacteria
69