27. Resuspend the beads in 1 mL of ice-cold IP buffer with salt (see
Subheading 2.2, item 13). Incubate at 4
C for 10 min in a
rotator at 20 rpm.
28. Immobilize the beads using a magnetic rack, incubate for
2 min, and discard the IP buffer with salt.
29. Resuspend the beads in 1 mL of ice-cold IP wash buffer and
incubate at 4
C for 10 min in a rotator at 20 rpm.
30. Immobilize the beads using a magnetic rack, wait for 2 min,
and discard the IP wash buffer (see Subheading 2.2, item 14).
31. Resuspend the beads in 1 mL ice-cold TE pH 8 and incubate at
4
C for 10 min in a rotator at 20 rpm.
32. Immobilize the beads using a magnetic rack, wait for 2 min,
and discard the TE buffer, repeat this step.
33. Resuspend the beads in 100 μL of 1Â Elution buffer and add
5 μL RNase A (10 mg/mL) and leave at RT for 30 min (this is
immunoprecipitated DNA).
34. At the same time, add 5 μL of RNase A (10 mg/mL) to the
previously saved 100 μL of input control chromatin sample
(from À20
C freezer, see step 19), and leave at RT for
30 min (see Note 10).
35. De-crosslink the RNase A-treated immunoprecipitated
(IP) DNA and input control chromatin, by heating at 65
C
in a water bath overnight.
36. Immobilize the beads using a magnetic rack, wait for 2 min,
and transfer the liquid which contains the immunoprecipitated
DNA to a fresh 1.5 mL microcentrifuge tube.
37. Resuspend the beads in 50 μL of TE pH 7.5 to wash any
remaining DNA from the beads.
38. Immobilize the beads using a magnetic rack, wait for 2 min,
and transfer the 50 μL of TE pH 7.5 to the immunoprecipitated DNA from step 36.
39. Add 50 μL of TE pH 7.5 to the input control DNA and every
sample should contain 150 μL.
40. Add 5 μL proteinase K (10 mg/mL) to each IP and input
control sample and incubate at 55
C in a water bath for 2 h,
to destroy all of the protein.
41. Purify the IP and input control DNA using a PCR Purification
Kit, elute in 20 μL of nuclease-free water.
42. Measure the concentration and assess the quality of the DNA
(see Notes 11 and 12). The DNA can then be used for ChIPseq or qPCR (see Note 13).
DNA Isolation from Mycobacteria
67
Subheading 2.2, item 13). Incubate at 4
C for 10 min in a
rotator at 20 rpm.
28. Immobilize the beads using a magnetic rack, incubate for
2 min, and discard the IP buffer with salt.
29. Resuspend the beads in 1 mL of ice-cold IP wash buffer and
incubate at 4
C for 10 min in a rotator at 20 rpm.
30. Immobilize the beads using a magnetic rack, wait for 2 min,
and discard the IP wash buffer (see Subheading 2.2, item 14).
31. Resuspend the beads in 1 mL ice-cold TE pH 8 and incubate at
4
C for 10 min in a rotator at 20 rpm.
32. Immobilize the beads using a magnetic rack, wait for 2 min,
and discard the TE buffer, repeat this step.
33. Resuspend the beads in 100 μL of 1Â Elution buffer and add
5 μL RNase A (10 mg/mL) and leave at RT for 30 min (this is
immunoprecipitated DNA).
34. At the same time, add 5 μL of RNase A (10 mg/mL) to the
previously saved 100 μL of input control chromatin sample
(from À20
C freezer, see step 19), and leave at RT for
30 min (see Note 10).
35. De-crosslink the RNase A-treated immunoprecipitated
(IP) DNA and input control chromatin, by heating at 65
C
in a water bath overnight.
36. Immobilize the beads using a magnetic rack, wait for 2 min,
and transfer the liquid which contains the immunoprecipitated
DNA to a fresh 1.5 mL microcentrifuge tube.
37. Resuspend the beads in 50 μL of TE pH 7.5 to wash any
remaining DNA from the beads.
38. Immobilize the beads using a magnetic rack, wait for 2 min,
and transfer the 50 μL of TE pH 7.5 to the immunoprecipitated DNA from step 36.
39. Add 50 μL of TE pH 7.5 to the input control DNA and every
sample should contain 150 μL.
40. Add 5 μL proteinase K (10 mg/mL) to each IP and input
control sample and incubate at 55
C in a water bath for 2 h,
to destroy all of the protein.
41. Purify the IP and input control DNA using a PCR Purification
Kit, elute in 20 μL of nuclease-free water.
42. Measure the concentration and assess the quality of the DNA
(see Notes 11 and 12). The DNA can then be used for ChIPseq or qPCR (see Note 13).
DNA Isolation from Mycobacteria
67