All the media were prepared in Milli RO grade water and autoclaved at 15 pounds per square inch for. 10 g 5g 5g

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1 }lppend~ STANDARD MEDIA All the media were prepared in Milli RO grade water and autoclaved at 15 pounds per square inch for 15 min unless otherwise indicated. LB Broth (Luria Bertani Broth) Bacto-Tryptone Bacto-Yeast extract The components were dissolved in 950 ml and the ph adjusted to 7.5 with 5 N NaOH and the volume adjusted to 1000 ml with. Agar at a concentration of 1.5 % 10 g 5g 5g was added whenever solid medium was required. Terrific Broth Bacto-Tryptone 12 g Bacto-Yeast extract 24 g 4ml After cooling, 100 ml sterile phosphate buffer was added. LB Broth with and Tween ml of glycerol and 2 ml of Tween-80 is added to 1 L of LB broth and sterilized. Lowenstein Jensen medium (modified) KH2P04 Magnesium sulphate Magnesium citrate L-asparagine Potato flour 2.40 g 0.24 g 0.60 g 3.60 g 12 ml 30 g 600ml The solution was sterilized, allowed to cool and 20 ml of Malachite green aqueous solution (20 %) was added. The solution was mixed with 1000 ml of homogenized whole eggs, filtered A-1

2 fi_ppendi{ through cotton gauze and dispensed in 50 ml cotton plugged tube (12 ml/tube). The slants were sterilized by incubation at 80 C for lhr for three consecutive days and then stored at 4 C. Middle brook (MB) 7H9 broth MB7H9 broth base Tween80 Middle brook (MB) 7H10 Agar MB7H10 agar base Santon's medium L-asparagine Citric acid K2HP04 MgS04 Ferric ammonium citrate 4.7 g 2ml 5ml 19 g 5ml 4.00 g 2.00 g 0.50 g 0.50 g 0.05 g 35ml Tween 80 2ml The components were dissolved; ph adjusted to 7.2 with 5 N NaOH and the volume made up to 1000 ml with. SOC medium Bacto-Tryptone 20 g Bacto-Yeast extract 5g 0.5 g KC1(250mM) 10ml The components were dissolved; ph adjusted to 7.2 with 5 N NaOH and the volume made up to 980 ml with. After sterilization, 20 ml of glucose (1M, 0.22J.L filter sterilized) was added. A-2

3 }lppendir_ REAGENTS FOR ACID FAST STAINING Carbol fuchsin (primary stain) Basic fuchsin Phenol Ethanol (96 %) 3g 5% 10ml Mixed 10 ml of Basic fuchsin to 90 ml of phenol and the solution was filtered through Whatman filter paper No. 1 Acid alcohol (decolorizer) HCI (cone.) Ethanol (96 %) Malachite green solution (counter stain) Malachite green 3ml 97ml 0.25 gin ANTIBIOTICS AND SUBSTRATES All antibiotic solutions were filter sterilized by a 0.22 f..lm filter (Millipore) and stored at -20 C for long-term use. Reagent Stock solution Final Cone. Final Cone. (in E. coli) (in Mycobacterium) Ampicillin 5 mg/ml in H f..lg/ml Kanamycin 5 mg/ml in H f..lg/ml 25 f..lg/ml Cycloheximide 5 mg/ml in H f..lg/ml 100 f..lg/ml Streptomycin 5 mg/ml in H f..lg/ml 15 f..lg/ml X-gal 40 mg/ml in DMF 40 f..lg/ml 40 f..lg/ml Ciprofloxacin 0.5 mg/ml in H f..lg/ml f..lg/ml REAGENTS AND BUFFERS All the reagents and buffers for DNA and protein work were prepared in Milli Q grade water and sterilized by autoclaving for 15 min at 15-psi pressure unless otherwise indicated. A-3

4 }lppendi{ A. Commonly used buffers i. Phosphate Buffered Saline (PBS), per liter KH2P04 K2HP04 ph ii. Tris HCl buffer 0.34 g 1.32 g 8.0 g 7.3 Tris-HCl buffer of desired strength was prepared by dissolving appropriate amount of Tris in distilled water and adjusting the ph with concentrated HCl..For bacteriological work 10 mm Tris- HCl (ph 8.0) was used. iii. Ethylene diamine tetra acetic acid (EDTA) 0.5 M solution of disodium salt of EDTA was prepared in Milli Q, ph adjusted to 8.0 with NaOH and stored at 4 C. iv. Normal Saline v. Tween Normal Saline 8.50 g 1000 ml (final volume) 0.02 %Tween -80 was added to normal saline. B. Reagents for genomic DNA isolation from Mycobacterium TE Buffer Tris-HCl (ph 8.0) EDTA Tris EDT A Saline (TES) Buffer Tris-HCl (ph 8.0) EDTA IOmM lmm lomm lmm 150mM A-4

5 Lysozyme Lysozyme 50 mg/ml in Milli Q (Store at -20 C} Proteinase K Proteinase K 20 mg/ml in Milli Q (Store at -20 C} Buffered Phenol Molten phenol containing 0.1 % 8-hydroxyquinoline was equilibrated with 1M Tris-HCl (ph 8.0) and twice with O.lM Tris-HCl (ph 8.0) till the ph> 7.8 and then it is stored submerged in 10 mm Tris-HCl (ph 8.0) in dark bottle at 4 C away from direct light. Chloroform: Isoamyl alcohol Solution contains 24 parts chloroform and 1 part Isoamyl alcohol. The solution is stored in dark bottles at 4 C. C. Buffers for plasmid isolation from E. coli i. Glucose Tris EDT A Buffer (GTE) Tris-HCl (ph 8.0) EDTA (ph 8.0) Glucose ii. NaOH-SDS Mix NaOH SDS iii. Acetate Mix 25mM lomm 50mM 0.2N 1.0% Solution contains 3 volumes of 3 M sodium acetate and 4 volumes of 7.5 M ammonium acetate. A-5

6 Jlppencf~ D. Electrophoresis buffers i. T AE Buffer (SOX) Tris Base Glacial Acetic Acid 0.5 M EDTA (ph 8.0) Final Volume 242g 57.1 m1 looml 1000 m1 ii. TBE Buffer (SX) Tris Base Boric Acid 0.5 MEDTA (ph 8.0) Final Volume iii. Tris-glycine (Tank buffer) Tris Base Glycine SDS Final Volume iv. lox Formaldehyde-Agarose gel buffer MOPS Sodium Acetate EDTA ph was adjusted to 7.0 with 10 N NaOH 54g 27.5 g 20ml 1000 ml 3.0 g 14.4 g 2.0 g 1000 ml 200mM 50mM lomm v. lx Formaldehyde Agarose gel running buffer lox Formaldehyde-Agarose gel buffer 37% Formaldehyde RNase free water E. Buffers for transformation 100 ml 20ml 880ml i. Transformation Buffer I (TFB I) MOPS Buffer (ph 7.0) RbCl 10mM 10mM A-6

7 )Ippendi:( ii. Transformation Buffer II (TFB II) MOPS Buffer (ph 6.5) RbCl CaCh F. Buffers for gel loading loomm lomm 50mM i. 6X dye for agarose gel elctrophoresis Bromophenol Blue Sucrose 0.25% 40% ii. Laemilli sample buffer (2X) SDS 2-mercaptoetllanol Bromophenol Blue Tris-HCl (ph 6.8) iii. SX RNA loading buffer Saturated Bromophenol blue 500 mm EDTA (ph 8.0) 37% Formaldehyde 100 % Formamide lox Formaldehyde-Agarose gel buffer4 ml 20% 4% 10% 0.2% 125mM 16 Ill 80 Ill 720 Ill 2ml 3084!!1 The volume was then made up to 10 ml with RNase free water. For loading onto tlle gel tlle buffer was added to a final concentration of IX to the RNA sample, the mixture incubated at 65 C for 3-5 min and chilled on ice. Before loading ofetbr (lmg/ml) was added to the sample. G. Reagents for protein induction.. i. Isopropyl P-D-thiogalactopyranoside (IPTG) 1 Min ii. Phenylmethylsulfonyl fluoride (PMSF) 100 mm in isopropanol A-7

8 Jlppendi:( H. Reagents for SDS-Polyacrylamide gel electrophoresis i. Acrylamide 30 /o Acrylamide N, N' -methylenebisacrylamide 29.2 g 0.8 g 50ml The solution was stirred to completely dissolve the acrylamide, the volume made up to 100 m1 and filtered through Whatman filter paper No. 1 ii. Ammonium per sulfate (APS) loo/o 1 g of APS in 10 m1 iii. SDS 20 /o 20 g SDS in 100 m1 iv. Buffer for resolving gel Tris-HCl (ph 8.8) 1.5 M v. Buffer for stacking gel Tris-HCl (ph 6.8) 0.5M vi. Coomassie blue staining solution Coomassie brilliant blue-r % Acetic Acid 10% Methanol 45% 45% The solution was filtered through Whatman filter paper No. 1 vii. Destaining solution Methanol Acetic acid 45% 10% 45% viii. Gel storage solution Acetic acid in 7% A-8

9 Jlppend~ I. Reagent used for Protein estimation i. Solution A Sodium carbonate Sodium potassium t~ate SDS lnnaoh 2.0 g 1.0 g 1.0 g loml The volume was made upto 100 m1 with distilled water and stored at room temperature. ii. Solution B CuS04.SH 2 0 iii. Solution C iv. Solution D 4% w/v in distilled water 100 part of Solution A and 1 part of Solution B were mixed Folin phenol reagent with water in 1: 1 proportion was mixed v. Bovine serum albumin (BSA) Dissolved bovine serum albumin (Sigma) in distilled water to the final concentration of 1 mg/ml. Stored at -20 C. A-9