19. In our hands, freezing overnight at À80
C was not essential
for M. tuberculosis genomic DNA extraction but significantly
increased genomic DNA extracted from NTM isolates.
20. Lysozyme treatment should turn the mycobacterial sample
turbid due to cell lysis.
21. Chloroform is a toxic, volatile solvent and should be worked
with a Class I fume extraction cabinet. All local working practices must be adhered to.
22. If it is not possible to incubate samples overnight at À20
C,
samples can be chilled for 30 min at À80
C.
23. The DNA may not always be visible as a pellet, but the DNA
yield may still be appropriate. Eluent choice is critical in DNA
extraction. TE buffer is recommended for the storage of
extracted DNA as it prevents degradation compared to storage
in water. However, TE buffer contains EDTA which is a metal
ion chelator. It can inhibit enzyme processes downstream,
including next-generation sequencing. Consider eluent choice
based on downstream applications when performing gDNA
extractions.
24. Inhibitors and contaminants introduced from procedure
reagents (e.g., CTAB, salts) or from samples (e.g., RNA, protein) can be carried over into the final eluted sample. It is
recommended that UV spectrometry (such as a NanoDrop
system) is used to assess purity, while a fluorometric-based
approach (e.g., Qubit) is suggested to quantify eluted DNA
samples. Ratio values of 280/260 and 260/230 can be used to
identify what is present in DNA extractions, see Table 1.
25. Alternatively, samples of genomic DNA can be run on a 0.7%
agarose gel at 75 for 1.5 h, run in 30 min extensions if necessary. There should be a high molecular weight band at the top
of the gel; smearing of the gel with an absence of this high
molecular weight band suggests gDNA is fragmented or
smearing could be RNA contamination. Gel electrophoresis
Table 1
UV spectrometry can be used to assess purity of DNA extractions [26]
Compound type
Absorbance (nm)
Ratio values (280/260)
Ratio values (260/230)
DNA
280
~1.8
2.0–2.2
RNA
280
~2.0
2.0–2.2
EDTA; salts
230
<1.5
<2.0
Carbohydrates
230
<1.5
<2.0
Phenol
230
<1.5
<2.0
DNA Isolation from Mycobacteria
73
C was not essential
for M. tuberculosis genomic DNA extraction but significantly
increased genomic DNA extracted from NTM isolates.
20. Lysozyme treatment should turn the mycobacterial sample
turbid due to cell lysis.
21. Chloroform is a toxic, volatile solvent and should be worked
with a Class I fume extraction cabinet. All local working practices must be adhered to.
22. If it is not possible to incubate samples overnight at À20
C,
samples can be chilled for 30 min at À80
C.
23. The DNA may not always be visible as a pellet, but the DNA
yield may still be appropriate. Eluent choice is critical in DNA
extraction. TE buffer is recommended for the storage of
extracted DNA as it prevents degradation compared to storage
in water. However, TE buffer contains EDTA which is a metal
ion chelator. It can inhibit enzyme processes downstream,
including next-generation sequencing. Consider eluent choice
based on downstream applications when performing gDNA
extractions.
24. Inhibitors and contaminants introduced from procedure
reagents (e.g., CTAB, salts) or from samples (e.g., RNA, protein) can be carried over into the final eluted sample. It is
recommended that UV spectrometry (such as a NanoDrop
system) is used to assess purity, while a fluorometric-based
approach (e.g., Qubit) is suggested to quantify eluted DNA
samples. Ratio values of 280/260 and 260/230 can be used to
identify what is present in DNA extractions, see Table 1.
25. Alternatively, samples of genomic DNA can be run on a 0.7%
agarose gel at 75 for 1.5 h, run in 30 min extensions if necessary. There should be a high molecular weight band at the top
of the gel; smearing of the gel with an absence of this high
molecular weight band suggests gDNA is fragmented or
smearing could be RNA contamination. Gel electrophoresis
Table 1
UV spectrometry can be used to assess purity of DNA extractions [26]
Compound type
Absorbance (nm)
Ratio values (280/260)
Ratio values (260/230)
DNA
280
~1.8
2.0–2.2
RNA
280
~2.0
2.0–2.2
EDTA; salts
230
<1.5
<2.0
Carbohydrates
230
<1.5
<2.0
Phenol
230
<1.5
<2.0
DNA Isolation from Mycobacteria
73