CTAB
George Odette
Abstract
Effective extraction and purification of DNA from mycobacteria is critical for downstream molecular applications. However, mycobacteria have a complex cell wall structure that makes DNA extraction challenging. Here, we describe an optimized protocol for extracting DNA from mycobacterial cultures grown on solid or in liquid media. Mycobacterial cells were harvested and underwent heat killing to inactivate nucleases. Cells were lysed using lysozyme, SDS, and proteinase K to break down the cell wall structure and release DNA. Contaminants were removed through extraction with chloroform/isoamyl alcohol and DNA was precipitated using isopropanol. Further washes with ethanol removed residual impurities. DNA yields from 32 extractions were quantified by quibit. The mean yield was 15.3 μg and the median 14 ng/ μl, with a range of 6-49 ng/ μl. The interquartile range was 11.5 to 18 ng/ μl. One outlier of 49 μg was observed. The chloroform extraction was critical to remove contaminating proteins, cell debris, and nucleases that could reduce DNA purity and yield. This protocol allows effective extraction of DNA from diverse mycobacterial species grown in either liquid or solid media. The DNA isolated can be used for downstream applications including PCR, restriction digestion, sequencing, and other molecular biology techniques.
Steps
Grow strains of interest on Löwenstein-Jensen medium at 37°C until growth becomes clearly visible
Transfer an appropriate number (1-3 inoculation loops) of bacterial cells into a microcentrifuge tube containing 400 μl TE-Puffer
Incubate for 20 min at 80° C in a water bath to kill bacteria (check temperature with thermometer as this seems to be crucial for DNA quantity and quality afterwards)
Centrifuge for 5 min at 13000 rcf, discard supernatant and add 400 μl TE-Puffer
Add 50 μl of 10mg/ml lysozym, vortex und incubate at least 1 h at 37° C or o/n (we always incubate over night)
Add 70 μl of 10% SDS and 10 μl proteinase, vortex shortly and incubate 1 hr at 60° C
Add 100 μl 5M NaCl
Add 100 μl CTAB/NaCl mix (prewarmed at 65°C), vortex until the liquid content becomes white and incubate 10 min at 65° C
Add approx. 750 μl chloroform/isoamyl alcohol mix (24:1), vortex 10 sec and centrifuge 15 min at 13000 rcf at RT
Transfer aqueous supernatant in a new tube
Add 0,6 volume (450 μl) isopropanol, mix carefully and incubate 30 min at -20°C
Centrifuge 15 min at 13000 g
Remove most of supernatant and add 500 μl of cold 70 % ethanol
Centrifuge for 15 min at ±11,000 xg.
Discard most of the supernatant; leave about 20 μL (3 mm height) above the pellet (see Note 4).
Add 1 mL of cold 70% ethanol (from the -20 °C freezer) and turn the tube a few times upside down to wash the DNA precipitate.
Centrifuge for 5 min at ±11,000 xg.
Discard most of the supernatant; leave about 20 μL (3 mm height) above the pellet (see Note 4).
Centrifuge for 1 min at ±11,000 xg.
Remove the remaining supernatant from above the pellet by pipetting very carefully with a 20μL pipette (see Note 15).
Permit the pellet to dry for ± 15 min at RT. Check whether all ethanol is evaporated. If not, then extend the drying time.
Dissolve the pellet in the amount of 1X TE estimated in step 10 (see Note 16).
