4. Place cuvette into the electroporation chamber and apply a
pulse at 2.5 kV, 1000 Ω, 25 μF (see Note 8).
5. Remove the cuvette from the chamber, recover the cells with a
sterile 1 mL disposable transfer pipet, and transfer the cells to
30 mL inkwell bottle containing 5 mL of 7H9-OADCTween 80.
6. Shake the cultures at 37
C for 1–2 days (see Note 9).
7. Plate dilutions of the culture on 7H10 plates containing
50 μg/mL hygromycin to obtain well-separated isolated colonies (see Note 10).
8. Allow the plates to dry, wrap them with 2 sheets of aluminum
foil (see Note 11), and incubate the plates at 37
C for 21 days.
3.3 SNP-Transfer
Identification
and Verification
1. Prepare a 24-well tissue-culture plate containing 1 mL of
7H9-OADC-tween 80 (50 μg/mL hygromycin) per well.
2. Pick 24 well-isolated hygromycin-resistant colonies from the
plates described in Subheading 3.2 (see step 7 in Subheading
3.2) and resuspend in the 24-well plate (one colony per well).
3. Cover the 24-well plate with sealing membrane to prevent
spillage and cross contamination. Wrap the plate with aluminum foil, place in a plastic container, and shake the cultures for
1 week at 37
C at 100 rpm, or until the cultures reach an
OD 600 of 1.0–1.5.
4. After a 5 days - 1 week incubation, mix the cultures in each well
with a 1 mL pipet to ensure uniform suspension; the OD 600
should be ~1.0–1.5 (but no more). Transfer 0.3 mL of the
cultures to screw-capped tubes and heat inactivate the cells by
total immersion of the tubes in hot metal bead bath (85
C) for
50 min (see Note 12).
5. Cover the plate with a new sealing membrane to prevent evaporation of the remainder of the cultures, which can then be
stored stationary at 37
C until the heat-killed cells are verified
by PCR screening and DNA sequencing.
Table 2
Selection for the lagging strand in M. tuberculosis sequences
Gene position
Gene orientation
Lagging strand (5
0 –3
0 )
a
0–2.2 Mb
Clockwise
Bottom strand
0–2.2 Mb
Counter-clockwise
Top strand
2.3–4.4 Mb
Clockwise
Top strand
2.3–4.4 Mb
Counter-clockwise
Bottom strand
a
Reading the dsDNA sequence file of the gene from left to right
Oligo-Mediated Recombineering in Mycobacteria
311
pulse at 2.5 kV, 1000 Ω, 25 μF (see Note 8).
5. Remove the cuvette from the chamber, recover the cells with a
sterile 1 mL disposable transfer pipet, and transfer the cells to
30 mL inkwell bottle containing 5 mL of 7H9-OADCTween 80.
6. Shake the cultures at 37
C for 1–2 days (see Note 9).
7. Plate dilutions of the culture on 7H10 plates containing
50 μg/mL hygromycin to obtain well-separated isolated colonies (see Note 10).
8. Allow the plates to dry, wrap them with 2 sheets of aluminum
foil (see Note 11), and incubate the plates at 37
C for 21 days.
3.3 SNP-Transfer
Identification
and Verification
1. Prepare a 24-well tissue-culture plate containing 1 mL of
7H9-OADC-tween 80 (50 μg/mL hygromycin) per well.
2. Pick 24 well-isolated hygromycin-resistant colonies from the
plates described in Subheading 3.2 (see step 7 in Subheading
3.2) and resuspend in the 24-well plate (one colony per well).
3. Cover the 24-well plate with sealing membrane to prevent
spillage and cross contamination. Wrap the plate with aluminum foil, place in a plastic container, and shake the cultures for
1 week at 37
C at 100 rpm, or until the cultures reach an
OD 600 of 1.0–1.5.
4. After a 5 days - 1 week incubation, mix the cultures in each well
with a 1 mL pipet to ensure uniform suspension; the OD 600
should be ~1.0–1.5 (but no more). Transfer 0.3 mL of the
cultures to screw-capped tubes and heat inactivate the cells by
total immersion of the tubes in hot metal bead bath (85
C) for
50 min (see Note 12).
5. Cover the plate with a new sealing membrane to prevent evaporation of the remainder of the cultures, which can then be
stored stationary at 37
C until the heat-killed cells are verified
by PCR screening and DNA sequencing.
Table 2
Selection for the lagging strand in M. tuberculosis sequences
Gene position
Gene orientation
Lagging strand (5
0 –3
0 )
a
0–2.2 Mb
Clockwise
Bottom strand
0–2.2 Mb
Counter-clockwise
Top strand
2.3–4.4 Mb
Clockwise
Top strand
2.3–4.4 Mb
Counter-clockwise
Bottom strand
a
Reading the dsDNA sequence file of the gene from left to right
Oligo-Mediated Recombineering in Mycobacteria
311