Isolation of Genomic DNA from Mycobacteria


Solutions

Lysozyme solution: 50 mg/ml.
Store in small aliquots at -20°C Use one aliquot each time, do not freeze and thaw twice.

10 %SDS
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.

5M NaCl

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.

Phenol/Chloroform
Mix 1 volume of Phenol (pH8.0 Tris HCl Saturated) with 1 volume of chloroform. Store at 4 °C, overnight.

Isopropanol

75% Ethanol

Procedure

  1. Inoculate a Löwenstein Jensen medium with the mycobacterial strain of interest and incubate at 37 °C until growth becomes clearly visible.
  2. 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.
  3. 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).
  4. Add 70 μl 10 % SDS, 10 μl proteinase K (20 mg/ml), vortex softly and incubate 15 min at 65 °C.
  5. Add 100 μl 5M NaCl.
  6. Add 100 μl CTAB/NaCl which is prewarmed at 65 °C. Vortex and incubate 10 min at 65° C.
  7. 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.
  8. 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.
  9. 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.
  10. 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 ).
  11. Redissolve the pellet in 100 μl TE buffer. 37 °C for 30 min or room temp until the DNA totally dissolved.

NOTES

  1. In procedure 3, cells should be separated enough before adding lysozyme.
  2. 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.
  3. In procedure 11, in order to dissolve DNA completely, the tube could be left in room temp or at 4 °C overnight.