Isolation of Genomic DNA from Mycobacteria
Lysozyme solution: 50 mg/ml.
Store in small aliquots at -20°C Use one aliquot each time, do not freeze and thaw twice.
10 g SDS/100 ml distilled water. Dissolve by heating at 65 °C for 20 min. Do not autoclave. Store at room temp for no longer than 1 month.
Proteinase K: 20 mg/ml.
Store in small aliquots at -20 °C Use one aliquot each time, do not freeze and thaw twice.
29.2 g NaCl/100 ml distilled water. Autoclave. Store at room temp for no longer than 1 year.
CTAB/NaCl (10 %CTAB in 0.7 M NaCl)
Dissolve 4.1 g NaCl in 80 ml distilled water. While stirring, add 10 g CTAB. If necessary, heat solution to 65 °C Adjust the volume to 100 ml with distilled water. Store at room temp. for no longer than 6 months.
Mix 1 volume of Phenol (pH8.0 Tris HCl Saturated) with 1 volume of chloroform. Store at 4 °C, overnight.
- Inoculate a Löwenstein Jensen medium with the mycobacterial strain of interest and incubate at 37 °C until growth becomes clearly visible.
- Transfer at least one loopfull of cells into a microcentrifuge tube (1.5 ml) containing 400 μl TE. Heat 30 min at 80 °C to kill cells. And cool at room temp.
- Add 4 to 5 glass beads, vortex to separate cells. Add 30 μl lysozyme（50 mg/ml) vortex and incubate at least 1 hour at 37 °C. (preferable overnight).
- Add 70 μl 10 % SDS, 10 μl proteinase K (20 mg/ml), vortex softly and incubate 15 min at 65 °C.
- Add 100 μl 5M NaCl.
- Add 100 μl CTAB/NaCl which is prewarmed at 65 °C. Vortex and incubate 10 min at 65° C.
- Add a volume of about 700 μl Phenol/Chloroform, turn the tube a few times upside down softly (in order not to break the DNA chain), and centrifuge at room temp for 15 min at 11,000 g. Transfer the aqueous supernatant to a new microcentrifuge tube carefully.
- Add the same volume of Phenol/Chloroform, turn the tube a few times upside down softly and centrifuge at room temp for 10 min at 13,000 g. Transfer the aqueous supernatant to a new microcentrifuge tube carefully.
- Add 0.6 volume isopropanol to precipitate the nucleic acids. Place at least 30 min at -20 °C (or longer), Spin 10 min at room temp in a microcentrifuge at 13,000 g.
- Discard the supernatant. Add 1 ml of cold 75 % Ethanol and turn the tube a few times upside down. Spin 5 min at room temp in a microcentrifuge and discard the supernatant cautiously. Permit the pellet to dry at room temp (about 10 min ).
- Redissolve the pellet in 100 μl TE buffer. 37 °C for 30 min or room temp until the DNA totally dissolved.
- In procedure 3, cells should be separated enough before adding lysozyme.
- In procedure 8, if the genomic DNA has been extracted good enough, white floccules could be seen. Using a pep tip, drag the floccules up and redissolve them in TE buffer.
- In procedure 11, in order to dissolve DNA completely, the tube could be left in room temp or at 4 °C overnight.