13. Add 100 μL of 5 M NaCl to each sample, then add 100 μL
CTAB/NaCl pre-warmed buffer. Mix gently by stirring the
pipette tip and incubate at 65
C for 15 min in a water bath.
14. Centrifuge the samples briefly, add 750 μL chloroform–isoamyl alcohol (24:1 v/v) solution and invert the samples ten
times to mix (see Note 22).
15. Centrifuge samples at 10,000 Â g for 5 min at room
temperature.
16. Label DNA LoBind 1.5 mL microcentrifuge tubes and add
450 μL À20
C (ice cold) 100% isopropanol in each tube.
17. Transfer the aqueous (upper) phase from centrifuged samples
to the LoBind microcentrifuge tubes containing ice-cold
isopropanol.
18. Invert samples ten times to mix, and store overnight at À20
C
to precipitate nucleic acid (see Note 23).
19. Centrifuge samples at 10,000 Â g for 15 min at room temperature. Remove as much supernatant as possible without damaging or disturbing pellets.
20. Wash the DNA pellet by adding 1 mL of ice-cold 70% ethanol
and invert ten times to mix.
21. Centrifuge samples at 10,000 Â g for 5 min at room temperature, discard supernatant and dry pellets, with the LoBind tube
lid open, in a heating block set at 65
C.
22. Rehydrate pellets with 50 μL Milli-Q water, and place at 4
C
overnight (see Note 24).
23. Assess DNA samples for purity and DNA concentration (see
Note 25) using a NanoDrop.
24. Store mycobacterial gDNA samples at À20
C.
4 Notes
1. All the methods for the handling of live M. tuberculosis should
be carried out in a BSC 1 in the CL3 laboratory. The centrifugation steps should be carried out in a centrifuge with aerosolcontainment rotors and screw cap tubes with O-rings for live
M. tuberculosis. Some steps require a Class I fume extraction
cabinet for the use of toxic, volatile reagents but the samples are
not infectious.
2. The electroporated cells with the desired plasmid are spread
onto 7H11 solid agar supplemented with 10% OADC and the
antibiotic of choice. Individual colonies are further subcultured
onto 7H11 solid agar supplemented with 10% OADC and the
antibiotic of choice. This reduces the background noise from
70
Heena Jagatia and Daire Cantillon
CTAB/NaCl pre-warmed buffer. Mix gently by stirring the
pipette tip and incubate at 65
C for 15 min in a water bath.
14. Centrifuge the samples briefly, add 750 μL chloroform–isoamyl alcohol (24:1 v/v) solution and invert the samples ten
times to mix (see Note 22).
15. Centrifuge samples at 10,000 Â g for 5 min at room
temperature.
16. Label DNA LoBind 1.5 mL microcentrifuge tubes and add
450 μL À20
C (ice cold) 100% isopropanol in each tube.
17. Transfer the aqueous (upper) phase from centrifuged samples
to the LoBind microcentrifuge tubes containing ice-cold
isopropanol.
18. Invert samples ten times to mix, and store overnight at À20
C
to precipitate nucleic acid (see Note 23).
19. Centrifuge samples at 10,000 Â g for 15 min at room temperature. Remove as much supernatant as possible without damaging or disturbing pellets.
20. Wash the DNA pellet by adding 1 mL of ice-cold 70% ethanol
and invert ten times to mix.
21. Centrifuge samples at 10,000 Â g for 5 min at room temperature, discard supernatant and dry pellets, with the LoBind tube
lid open, in a heating block set at 65
C.
22. Rehydrate pellets with 50 μL Milli-Q water, and place at 4
C
overnight (see Note 24).
23. Assess DNA samples for purity and DNA concentration (see
Note 25) using a NanoDrop.
24. Store mycobacterial gDNA samples at À20
C.
4 Notes
1. All the methods for the handling of live M. tuberculosis should
be carried out in a BSC 1 in the CL3 laboratory. The centrifugation steps should be carried out in a centrifuge with aerosolcontainment rotors and screw cap tubes with O-rings for live
M. tuberculosis. Some steps require a Class I fume extraction
cabinet for the use of toxic, volatile reagents but the samples are
not infectious.
2. The electroporated cells with the desired plasmid are spread
onto 7H11 solid agar supplemented with 10% OADC and the
antibiotic of choice. Individual colonies are further subcultured
onto 7H11 solid agar supplemented with 10% OADC and the
antibiotic of choice. This reduces the background noise from
70
Heena Jagatia and Daire Cantillon