5 Notes
1. Preparing Complete 7H9 Media.
(a) Suspend 2.35 g of Middlebrook 7H9 media in 450 ml
water and add 1 mL glycerol and 0.25 polysorbate 80.
(b) Heat and mix to dissolve the media completely.
(c) Sterilize media by autoclaving at 120
C for 10 min.
(d) Cool to below 45
C and aseptically add 25 mL Middlebrook OADC growth supplement.
(e) Mix well before using.
2. Preparing Complete 7H10 Solid Media.
(a) Suspend 9.73 g of Middlebrook 7H10 media in 450 mL
water and add 2.5 mL glycerol.
(b) Repeat steps b–e of Note 1.
(c) Aseptically pour 5 mL of the complete 7H10 media into
agar plates and leave to cool and solidify at room
temperature.
(d) Store agar plates at 4
C until needed.
3. Denaturation Buffer.
6 M urea, 2 M thiourea in 10 mM tris buffer pH 8.0. (It’s
good practice to make this fresh.)
4. Preparation of Stage Tips.
(a) Use sharp forceps to make a hole in the center of the lid of
a 2 mL Eppendorf tube.
(b) Insert a 200 μL pipette tip.
(c) Cut a disk of C18 resin (0.4–0.5 mm) with “cookie cutter” apparatus.
(d) Eject the disk into the yellow stage tip by pushing it out
with a wire.
Table 1
Summary statistics obtained through MaxQuant suite software, showing obtained MS/MS spectra,
peptide and proteins with their calculated FDR and missed cleavages
Summary statistics
Count
Spectra identified
52,520
Unique peptides identified
7805
Proteins identified
1387
Peptide FDR
0.00205
Protein FDR
0.009373
Missed cleavages
0 (81%), 1 (18%), 2 (1%)
186
John Iradukunda et al.
1. Preparing Complete 7H9 Media.
(a) Suspend 2.35 g of Middlebrook 7H9 media in 450 ml
water and add 1 mL glycerol and 0.25 polysorbate 80.
(b) Heat and mix to dissolve the media completely.
(c) Sterilize media by autoclaving at 120
C for 10 min.
(d) Cool to below 45
C and aseptically add 25 mL Middlebrook OADC growth supplement.
(e) Mix well before using.
2. Preparing Complete 7H10 Solid Media.
(a) Suspend 9.73 g of Middlebrook 7H10 media in 450 mL
water and add 2.5 mL glycerol.
(b) Repeat steps b–e of Note 1.
(c) Aseptically pour 5 mL of the complete 7H10 media into
agar plates and leave to cool and solidify at room
temperature.
(d) Store agar plates at 4
C until needed.
3. Denaturation Buffer.
6 M urea, 2 M thiourea in 10 mM tris buffer pH 8.0. (It’s
good practice to make this fresh.)
4. Preparation of Stage Tips.
(a) Use sharp forceps to make a hole in the center of the lid of
a 2 mL Eppendorf tube.
(b) Insert a 200 μL pipette tip.
(c) Cut a disk of C18 resin (0.4–0.5 mm) with “cookie cutter” apparatus.
(d) Eject the disk into the yellow stage tip by pushing it out
with a wire.
Table 1
Summary statistics obtained through MaxQuant suite software, showing obtained MS/MS spectra,
peptide and proteins with their calculated FDR and missed cleavages
Summary statistics
Count
Spectra identified
52,520
Unique peptides identified
7805
Proteins identified
1387
Peptide FDR
0.00205
Protein FDR
0.009373
Missed cleavages
0 (81%), 1 (18%), 2 (1%)
186
John Iradukunda et al.