9. It is important to keep this spent liquid in case the antibody
does not work for the protein of interest. Additionally, the
specificity of the antibody should be tested prior to the commencement of the ChIP experiment.
10. The input control DNA is very important for the sequencing
steps, as it shows the specificity of the antibody. During
sequencing, you will see many nonspecific peaks before the
addition of the antibody to the sample; therefore, the addition
of the antibody specific for the protein of interest will minimize
background noise. It is also very important to treat the IP and
input control DNA exactly the same at the latter end of the
immunoprecipitation steps for this reason.
11. The DNA concentration can range between 0.1 and 0.5 ng/μL
for immunoprecipitated (IP) DNA and 10 and 25 ng/μL as
determined using qubit spectrophotometer; however, this
largely depends on the antibody used. Importantly check the
purity using the A260/A280 ratio which should be 1.7–1.9.
12. The DNA concentration for the input control DNA will be
considerably higher than the IP DNA, this is because the input
control DNA takes the whole genome into consideration while
the IP sample represents only DNA crosslinked to the protein
of interest.
13. qPCR for gene targets of the protein of interest can be implemented before sending DNA samples for sequencing as an
indicator of the success of the immunoprecipitation.
14. Steps 17–24 to isolate genomic DNA for qPCR from
M. tuberculosis can be performed outside of the CL3
laboratory.
15. The restriction digested DNA should appear as multiple bands
on the agarose gel, this is an indicator for the quality of the
extracted DNA. 10 mL culture of M. tuberculosis can yield
~3–4 μg/μL DNA.
16. All NTM handling should take place in containment level
2 (CL2) laboratories.
17. M. abscessus clinical isolates may grow poorly if biomass is taken
from agar plates and used to inoculate 7H9 broth. If this is the
case, passage this agar biomass into 10 mL 7H9 broth and
incubate for 10 days at 37
C, 180 rpm. Subculture 5 mL into
20 mL 7H9 broth and incubate at 37
C until culture becomes
turbid. If culturing NTM isolates for the first time, incubate in
duplicate with one culture at 30
C and one culture at 37
C to
determine best incubation temperature [25].
18. TE buffer is used here, not molecular-grade water, to act as a
suitable buffer for subsequent lysozyme treatment
downstream.
72
Heena Jagatia and Daire Cantillon
does not work for the protein of interest. Additionally, the
specificity of the antibody should be tested prior to the commencement of the ChIP experiment.
10. The input control DNA is very important for the sequencing
steps, as it shows the specificity of the antibody. During
sequencing, you will see many nonspecific peaks before the
addition of the antibody to the sample; therefore, the addition
of the antibody specific for the protein of interest will minimize
background noise. It is also very important to treat the IP and
input control DNA exactly the same at the latter end of the
immunoprecipitation steps for this reason.
11. The DNA concentration can range between 0.1 and 0.5 ng/μL
for immunoprecipitated (IP) DNA and 10 and 25 ng/μL as
determined using qubit spectrophotometer; however, this
largely depends on the antibody used. Importantly check the
purity using the A260/A280 ratio which should be 1.7–1.9.
12. The DNA concentration for the input control DNA will be
considerably higher than the IP DNA, this is because the input
control DNA takes the whole genome into consideration while
the IP sample represents only DNA crosslinked to the protein
of interest.
13. qPCR for gene targets of the protein of interest can be implemented before sending DNA samples for sequencing as an
indicator of the success of the immunoprecipitation.
14. Steps 17–24 to isolate genomic DNA for qPCR from
M. tuberculosis can be performed outside of the CL3
laboratory.
15. The restriction digested DNA should appear as multiple bands
on the agarose gel, this is an indicator for the quality of the
extracted DNA. 10 mL culture of M. tuberculosis can yield
~3–4 μg/μL DNA.
16. All NTM handling should take place in containment level
2 (CL2) laboratories.
17. M. abscessus clinical isolates may grow poorly if biomass is taken
from agar plates and used to inoculate 7H9 broth. If this is the
case, passage this agar biomass into 10 mL 7H9 broth and
incubate for 10 days at 37
C, 180 rpm. Subculture 5 mL into
20 mL 7H9 broth and incubate at 37
C until culture becomes
turbid. If culturing NTM isolates for the first time, incubate in
duplicate with one culture at 30
C and one culture at 37
C to
determine best incubation temperature [25].
18. TE buffer is used here, not molecular-grade water, to act as a
suitable buffer for subsequent lysozyme treatment
downstream.
72
Heena Jagatia and Daire Cantillon