i. Sauton's medium (Sauton, 1912) L-asparagine monohydrate 4.00 g Citric acid K2HP04 MgS04 Ferric ammonium citrate Glycerol Tween 80 DW

Transcription

1 1 Media Media for mycobacterial culture were prepared according to the description laid down by the International Union oftuberculosis (IUT) (Long,l958). i. Sauton's medium (Sauton, 1912) L-asparagine monohydrate 4.00 g Citric acid K2HP04 MgS04 Ferric ammonium citrate Tween g 0.50 g 0.50 g 0.05 g 35 ml 2 ml 900ml The components were dissolved; its ph adjusted to 7.2 with ION NaOH and the volume made up to IOOOml with. ii. Middle brook (MB) 7H9 broth MB7H9 broth base 4.7 g Tween80 2 ml 5 ml 900ml iii. Middle brook (MB) 7H10 Agar MB7H10 agar base 19 g Tween 80 iv. Lowenstein Jensen medium Mineral salt solution KH2P04 Magnesium sulphate Magnesium citrate L-asparagine 2 ml 5 ml 900 ml 0.4% 0.04% 0.01% 0.6% 2% All salts were dissolved in distilled water. AI

2 To prepare L-J medium, 300ml of mineral salt solution was mixed with 600ml of egg fluid by beating the egg. This was followed by addition of 12 ml of aqueous solution of malachite green (2%). The suspension was mixed and filtered with musilin cloth before distribution into tubes. The tubes were sterilized in slanting position at 80 C for 1 hr daily for 3 consecutive days. v. Luria Bertani Broth (LB Broth ) (Sambrook et al., 1989) Bacto-Tryptone 10 g Bacto-Yeast extract 5 g 5g The components were dissolved in 950ml and the ph adjusted to 7.5, then volume made up to 1 OOOml. vi. Luria Bertani Broth (LB) agar: LB agar was prepared by adding 1.2% of agar powder into LB broth before autoclaving. vn. LB Broth with and Tween- 80 5ml of glycerol and 2ml oftween-80 is added to 1L oflb broth and sterilized viii. SOC medium (Sambrook et. al., 2001) Bacto-Tryptone 20 g Bacto-Yeast extract KCl (250mM) 5g 0.5 g 10 ml 900ml The components were dissolved; its ph adjusted to 7.2 with 5N NaOH and the volume made up to 980ml with. After sterilization, 20ml of glucose (1M, filter steri lized) was added. ix. Hertmans-de Boot minimal broth (HdeB) media. EDTA MgClz CaC12 6H20 NaMo04.2H20 CaClz O.Olg/L 0.1g/L 0.4g/L 0.4g/L A2

3 MnCh 2H20 lmg/l ZnS04?H20 2mg/L FeS04?H20 5mg/L CuS04 5H mg/l Supplemented with 15 mm ammonium sulphate, 16 mm phosphorous and 0.2% Tween 80. x. Meidia for Tissue culture RPMimedium RPMI HE PES NaHC03 1 pack 5.96 g 2.00 g The media was filtered through filter and 10% Fetal Bovine Serum (FBS) was added at the time of use 2. Reagents for acid fast staining i. Carbolfuchsin (primary stain) Basic fuchsin 3 g Phenol Ethanol (96 %) 5% loml Mixed 10 ml ofbasic fuchsinto 90 ml of Phenol and the solution was filtered through Whatman filter paper No. 1 ii. Acid alcohol (decolorizer) HCl (cone.) Ethanol (96 %) 3 ml 97 ml iii. Malachite green solution (counter stain) Malachite green 0.25 gin A3

4 3. Antibiotics and substrates All antibiotic solutions were filter sterilized by a 0.22!-l filter (Millipore) and stored at 20 C for long-term use. Reagent Ampicillin Kanamycin Cyclohexamide Chloramphenicol Streptomycin X-gal Nalidixic Acid. - Stock solution 5mg/ml in H20 5mg/ml in H20 5mg/ml in H20 34mg/ml in ethanol 5mg/ml in H20 40mg/ml in DMF 5mg/ml in H20 Final Cone. Final Cone. (in E. coli) (in Mycobacterium) 50 J.lg/ml - 50 J.lg/ml 25 J.lg/ml 100 J.lg/ml 100 J.lg/ml 34 J.lglml - 50 J.lg/ml 15J.!glml 40 J.lglml 40 J.lglml 50 J.lglml - 4. Commonly used reagents and buffers A. Buffers i. Phosphate Buffered Saline (PBS) KCl Na 2 HP g 0.20 g 1.44 g 900ml The components were dissolved, its ph adjusted to 7.4 and the volume made up to 1000ml. ii. Ethylene diamine tetra acetic acid (EDT A) 0.5 M solution of disodium salt of EDTA was prepared in Milli Q, ph adjusted to 8.0 with NaOH pellets and stored at 4 C. iii Tris HCl buffer 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 10mM Tris-HCl (ph 8.0) was used. A4

5 iv. Normal Saline 8.50 g 1000 ml v. Tween Normal Saline 0.02% Tween 80 was added to normal saline. B. Buffers for plasmid isolation from E. coli i. Glucose Tris EDTA Buffer (GTE) Tris-HCl (ph 8.0) 25 mm EDTA (ph 8.0) Glucose ii. NaOH-SDS Mix NaOH SDS 10 mm 50mM 0.2N 1.0% iii.acetate Mix Solution contains 3 volumes of 3 M sodium acetate and 4 volumes of 7.5 M ammonium acetate. C. Reagents for genomic DNA isolation from Mycobacteria i. TE Buffer Tris-HCl (ph 8.0) EDTA 10mM 1mM ii. Tris EDT A Saline (TES) Buffer Tris-HCl (ph 8.0) 10 mm EDTA lmm 150mM A5

6 iii. Lysozyme Lysozyme 50 mg/ml in (Store at -20C) iv. Proteinase K Proteinase K 20 mg/ml in (Store at -20C) v. 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 lomm Tris-HCl (ph 8.0) in dark bottles at 4 C away from direct light. vi. Chloroform: Isoamyl alcohol Solution contains 24 parts chloroform and 1 part Isoamyl alcohol. The solution is stored in light-tight glass bottles at 4 C. C. Electrophoresis Buffers i. TAE Buffer (SOX) Tris Base Glacial Acetic Acid 242 g 57.1 ml 0.5M EDTA (ph 8.0) 100 ml Final Volume 1000 ml ii. TBE Buffer (SX) Tris Base Boric Acid 54 g 27.5 g 0.5M EDTA (ph 8.0) 20 ml Final Volume 1000 ml iii. Tris-glycine (Tank buffer) Tris Base 3.0 g Glycine 14.4g SDS 2.0g A6

7 Final Volume 1000 ml D. Buffers for transformation i. Transformation Buffer I (TFB I) MOPS Buffer (ph 7.0) 10 mm RbCl 10mM ii. Transformation Buffer II (TFB II) MOPS Buffer (ph 6.5) 100 mm RbCl CaCh C. Buffers for gel loading 10mM 50mM i.6x dye for agarose gel elctrophoresis Bromophenol Blue 0.25 % Sucrose 40% ii. Laemmli sample buffer (2X) 20 % SDS 2-mercaptoethanol Bromophenol Blue Tris-HCl (ph 6.8) 4% 10% 0.2% D. Reagents for southern hybridization 125mM i. Denaturation buffer: NaOH 0.2 M, NaCI 1.5 M. ii. Neutralization buffer: m. 2ox sse: Tris-HCL (ph8.0) 1.0M, 1.5M 175.3g and Na citrate 88.2g. Volume was made uptoloooml, after adjusting ph 7.0 with 10 N NaOH. iv. Prehybridization buffer: 5X SSC, 10% N-lauryl sarcosine, 0.02 % SDS, 1% blocking buffer A7

8 v. Hybridization solution: vi.washing solution: a.genius buffer 1 ( GBl): b.genius buffer 2 ( GB2): Prehybridization solution containing probe. Tris- HCL (ph7.5) 100mM, NaC1150 mm. Genius buffer 1 containing 2 % blocking reagent. MgCh 50 mm. c.genius buffer 3 ( GB3): vi. Devoloping solution (10~1) : Tris- HCl (ph 9.5) 100mM, NaC1100 mm, NBT (stock 50mg/ml) 66 fll, BCIP (stock 50mg/ml) 33fll E. Reagents for protein induction i. Isopropyl P-D-thiogalactopyranoside (IPTG) 1 Min F Phenylmethylsulfonyl fluoride (PMSF) 10 mm in isopropanol G. Reagents for SDS-Polyacrylamide gel electrophoresis i. Acrylamide 30 % Acrylamide 29.2 g N, N' -methylenebisacrylamide 0.8 g 50ml The solution was stirred to completely dissolve the acrylamide, the volume made up to 100 ml and filtered through Whatman filter paper No. 1 ii. Ammonium per sulfate (APS) 10% 1 g of APS in 10 ml A8

9 iii. SDS 20% iv.buffer for resolving 1.5 M Tris-HCl (ph 8.8) v. Buffer for stacking gel 0.5 M,Tris-HCl (ph 6.8) 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 45 % Acetic acid 1 0 % 45% Gel storage solution Acetic acid 7 % H. Reagents for Western Blot analysis i. Transfer buffer Tris Glycine Methanol SDS M M 20% 0.1% ii.tris-buffered Saline (TBS) Tris-HCl (ph 7.5) 20mM 50mM A9

10 iii. Blocking Buffer TBS containing 0.5 % Tween 20 iv. Antibody conjugate solution Anti-His HRP conjugate diluted in washing buffer in dilutions according to manufacturers instructions. v. Washing Buffer TBS containing 0.05 % Tween 20 vi.developing Solution Tris-HCl (ph 7.5) 50 mm HzOz Diaminobenzidine(D AB) 18 ml 20 f.!l 12 mg I. Buffers for protein purification with His Bind resin i. Binding buffer Tris-HCl (ph 7.5) 20mM Imidazole 200mM 5mM 15% ii. Wash buffer Tris-HCl (ph 7.5) Imidazole 20mM 200mM 60mM 15% AIO

11 iii. Elution buffer Tris-HCl (ph 7.5) Imidazole 20mM 200mM 1M 15% iv. Strip buffer Tris-HCl (ph 7.5) EDTA 20mM 200mM loomm v. Charge buffer NiS04 J. Reagent used for Protein estimation 50mM i. Solution A Sodium Carbonate Sodium potassium tartarate SDS 1NNaOH 2.0 g 1.0 g 1.0 g loml The volume was made up to 1 OOml with distilled water and stored at room temperature. ii. Solution B 4% w/v in distilled water iii. Solution C 100 part of Solution A and 1 part of Solution B were mixed iv. Solution D 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 10mg/ml. Stored at -20C.

12 vi. Congo red Stock solution 5mg/ ml prepared in autoclaved distilled wated and the filtered through 0.45 JlM syringe filter before use. K.Reagents for osmotic shock i. Hypertonic solution ii. Hypotonic solution 20 mm Tris-HCl (ph 7.5) 20 % sucrose, and 0.5 mm EDTA 20 mm Tris-HCl (ph 7.5) L.Reagents for TLC i. Methanol ii. Choloroform A 12