Linearity

Enzyme production (EP) medium

Mycelial characteristicsof pathogen

Identificationof bioagent

Isolation and purification of Trichoderma sp.

Isolation and purification of pathogen, Fusarium udum

Isolation, purification and morphological characterization of pathogen and bioagent

Production of chitinase

Resistance to ampicillin

Citrate utilisation tes

Nitrate reduction test

Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test

Urease production

Gelatinase production

Growth at different temperatures

Salt tolerance tes

Voges Proskauer (acetoin production) test

Indole production

Carbon source utilisation tes

Carbohydrate fermentation test

Amino acids utilisation test (Decarboxylase/dihydrol

Species level identification

Urease productio

Gelatinase productio

Growth at different temperature

Salt tolerance tes

Voges Proskauer (acetoin production) tes

Indole production

Carbon source utilisation t

Carbohydrate fermentation tes

Amino acids utilisation test (Decarboxylase/dihydrola

Species level identification

Post-challenge survival

2D QSAR

Secondary antibodies

Primary antibodies

Antibodies used in our study

GFP-β-catenin-C-N (151-423 a.a) (ARM-N)

Antibodies

Irradiation of bacteria

ELISA blocking buffer

Lymphoproliferation

Resuspension solution

T-Cell Receptor Mutation Assay

NP-40 1% Tris 50 mM Sodiun deoxycholate 0.5% SDS 0.1% pH adjusted to 8.0 Running buffer for Western blotting Glycine 14.4g/l Tris base 3.05g/l SDS 1.0g/l Transfer buffer for western blotting Glycine 14.4g/l Tris base 3.03g/l The above salts were dissolved in 800ml of miliQ water and 200ml of methanol was then added. The buffer was chilled before use. PBST for Western blot 10X PBS (1000 ml) Sodium chloride 80 g Potassium chloride 2 g Disodium hydrogen phosphate 14.1 g (Na2HPO4) Potassium dihydrogen phosphate 2.49 g (KH2PO4) 1 l of 1X PBS + 1 ml of Tween-20

TBF-I buffer (200ml) Potassium acetate 0.588 g Calcium chloride 0.249 g Manganese chloride 1.98 g Rubidium chloride 2.418 g 15% Glycerol 30 ml pH adjusted to 5.8 with 1M acetic acid TBF-II buffer (100 ml) MOPS 0.209 g Calcium chloride 1.102 g Rubidium chloride 0.120 g 15% Glycerol 15 ml pH adjusted to 6.5 with 1M potassium hydroxide Acrylamide solution (30%) Acrylamide 29 g Bis-acrylamide 1 g H2O 100 ml Non denaturing polyacrylamide gel (12%) 30% acrylamide 38.6 ml H2O 40.6 ml TBE 20 ml 10% APS 0.7 ml RIPA buffer (Radio Immuno Precipitation Assay buffer): RIPA buffer for bacterial cell lysis Sodium chloride 150 mM

Water to 1000 ml MacConkey lactose agar: MacConkey Agar Base (Difco) 51.5 g Lactose 1% Water to 1000 ml Maloy agar: Tryptone 5 g Yeast extract 5 g NaCl 10 g NaH2PO4 10 g Chlorotetracycline (12.5 mg/ml) 4 ml Water 1000 ml Bacto-agar 15 g After autoclaving, the following solutions were added, ZnCl2 (20 mM) 5 ml Quinaldic acid (10 mg/ml) 10 ml Citrate buffer: (0.1 M; pH 5.5) Citric acid (0.1 M) 4.7 volumes Sodium citrate (0.1 M) 15.4 volumes TBE and TAE buffers: TBE: 90 mM Tris-borate, 2 mM EDTA (pH 8.0) and TAE: 40 mM Tris-acetate, 2 mM EDTA (pH 8.0) were used as standard electrophoresis buffers. TBE and TAE were prepared as 10X and 50X concentrated stock solutions, respectively, and used at 1X concentration

cto-agar 15 g LBON agar: LBON medium1000 mlBacto-agar 15 gLB soft agar: LB medium 100 ml Bacto-agar 0.6 gK-Medium: KH2PO4 1.0 mM FeSO4 0.5 mg/l (NH4)2SO4 1.5 mM MgCl2 0.08 mM Casamino acids 5 g/l Thiamine 2 mg/l pH was adjusted to 7.0 with Tris free base. K-medium is low osmolarity (70 mOsm) medium (Kennedy, 1982). Z broth: LB medium 100 ml CaCl2 (0.5 M) 0.5 ml MacConkey agar: MacConkey Agar (Difco) 51.5 g Water to 1000 ml MacConkey galactose agar: MacConkey Agar Base (Difco) 51.5 g Galactose 1%

Glucose/Glycerol-minimal A 19 amino acid medium: This medium is essentially the same as glucose/glycerol-minimal A medium described above except that all the 19 amino acids (except tryptophan) were added after autoclaving in a final concentration of 40 μg/ml from autoclaved 4mg/ml amino acid stock solutions. Minimal agar: Contained 1.5% Bacto-agar (Difco) in minimal A Medium. The plates were poured after mixing double strength minimal A medium with 4% agar (in water) that had been autoclaved separately. Wherever required, to test polaity relif at lacZ(am) or trpE(fs), meliobose (0.2%) was replaced for glucose and anthranilate at 100 μg/ml (4 mg/ml stock prepared in DMF) was replaced for tryptophan respectively. LB medium: Tryptone 10 g Yeast extract 5 g NaCl 10 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH. LBON medium: Tryptone 10 g Yeast extract 5 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH LB agar: LB medium 1000 ml

All the media and buffers were sterilized by autoclaving for 15 minutes at 121°C. Media and buffers used in this study are described below. Glucose/Glycerol-minimal A medium: K2HPO4 10.5 g KH2PO4 4.5 g (NH4)2SO4 1 g Sodium citrate, 2H2O 0.5 g Water to 1000 ml After autoclaving the following solutions were added. MgSO4 (1 M) 1 ml Glucose (20%) 10 ml Or Glycerol (80%) 5ml Vitamin B1 (1%) 0.1 ml Amino acids and bases, when required, were added to a final concentration of 40 μg/ml. When growth on other carbon sources was to be tested, glucose was substituted by the appropriate sugar at 0.2%; when used as carbon source, the final concentration of Casamino acids was 0.5%

Media and buffers

Antibiotics were used at the following final concentrations in various media as given inTable 2.4.Table 2.4Concentrations of antibiotics (μg/ml)

Antibiotics

Spheroplast resuspension buffer0.1M KCl15 mM HEPES (pH 7.5)3 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA)10% GlycerolPhosphatidylinositol sonication buffer10 mM HEPES (pH 7.5)1 mM EGTA PI3-kinase reaction buffer40 mM HEPES (pH 7.5)20 mM MgCl280 μM ATP5 μCi γ-P32ATPDeveloping solution for thin layer chromatography(120.2 ml)Chloroform –60 mlMethanol –47 mlAmmonia –4.4 mlWater –8.8 ml

Reagents for PI3-kinase assay

SDS-loading buffer was prepared as a 4X stock solutionin H2Oand used at a 1X concentration.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSRunning buffer was preparedas a 10X stock solution and diluted to 1X concentration before use.Buffers for Western blotanalysisTransfer buffer (10X stock solution)0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSTransfer buffer was prepared as a 10X stock solution and diluted to 1X concentration.1X Transfer buffer (1 litre)200 ml of methanol100 ml of 10X transfer buffer700 ml of waterTris-BufferSaline (TBS)25 mM Tris150 mM NaClpH was adjusted to 7.4 with HCl.TBS buffer was prepared asa10X stock solution and diluted to 1X concentration.Blocking and wash buffers (PBS-T and TBS-T)5% Fat-free milk0.1% Tween-20Volume was made to 100 ml with 1X TBS

1 mM sodium orthovanadate1 X protease inhibitor cocktail SDS-PAGE30% Acrylamide solution29 g Acrylamide1 gBis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OResolving gel mix (12%) (20 ml)6.6 ml H2O8 ml 30% acrylamide:bisacrylamide (29:1) mix5 ml 1.5 M Tris-HCl (pH 8.8)200 μl 10% SDS200 μl 10% Ammonium persulfate(APS)8 μl N,N,N′,N′-Tetramethylethylenediamine(TEMED)Stacking gel mix (5%, 6 ml)4.1 ml H2O1 ml 30% acrylamide:bisacrylamide (29:1) mix750 μl 1 M Tris-HCl (pH 6.8)60 μl 10% SDS60 μl 10% APS6 μl TEMEDSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol Blue2% DTT

Whole cell lysis buffer (Homogenizing buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM sodium fluoride

Buffers for protein extraction and analysis by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 MSorbitol50 mM β-mercaptoethanol (To be added just before use)Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSAE buffer3 M Sodium acetate(pH 5.3)0.5 M EDTA (pH 8.0)Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated Phenol24 ml Chloroform1 ml Isoamyl alcholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollDNA sample loading buffer was prepared in water

Buffer A50 mM Tris-HCl(pH 8)10 mM EDTA

Buffers for extraction and analysis of genomic DNA and RNA

Stripping solutionfor DNA1% SDS0.1% SSCDesired volume was adjusted with sterile water. Alternatively, 0.4 M NaOH was also used to stripthe bound probes fromnylon membranes.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESpH was adjusted to 7.5 with NaOH.HEPES was used as a buffering agent for preparing plates of YNB medium of different pH. Buffer was filter-sterilized and stored in an amber-coloured bottle.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OpH was adjusted to 6.7 with 1 N KOH.Yeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/ml carrier DNADimethyl sulfoxide (DMSO)Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml Zymolyase1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

pH was adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared asa50Xstock solution and used at 0.5X concentration.Alkaline denaturing solution for DNAfor membrane preparation0.5 M NaCl0.25 M NaOHVolume was adjusted with sterile water.Denhardt’s solution (50X)1%Ficoll-4001% Polyvinyl pyrollidone1% Bovine serum albuminVolume was adjusted with water and solution was stored at -20°C.Saline Sodium Citrate (SSC) buffer(20X)3.0 M Sodium chloride0.3 M Sodium citrate Volume was adjusted with water and solution was sterilized by autoclaving.Prehybridization Buffer5X SSC5X Denhardt’s solution50% Filtered formamide1% SDSVolume was adjusted with sterile water.Post hybridization wash buffersWash buffer 12X SSC0.1% SDSWash buffer21X SSC0.1% SDS

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving.PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10Xstock solution and used at a 1X concentration.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATris-Acetic acid EDTA (TAE) buffer40 mM Tris base0.5 M EDTA

Common buffers

Buffers and solutions

Binding Buffer (10X)

EMSA Buffer

Nuclear lysis buffer

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

For Electrophoretic mobility shift assay (EMSA)

TBST

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

For preparation of Ultra competent cells

Inoue buffer

(f) Running buffer

0.83mL1.5 M Tris-HCl,pH 6.8 50μL10% SDS 50μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)Resolving gel mix (12%) (20 ml)6.6 mLH2O 8 mL 30% acrylamide:bisacrylamide (29:1) mix 5 mL1.5 M Tris-HCl,pH 8.8 200 μL10% SDS 200 μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)

Whole cell lysis buffer for yeast (Homogenizing buffer) 50 mM Tris-HCl,pH 7.52 mM EDTA yeastprotease inhibitor cocktail SDS-PAGE 30% Acrylamide solution 29 g acrylamide 1 g bis-acrylamide dissolved in 100 mLH2O. 10% sodium dodecyl sulfate (SDS) 10 g SDS in 100 mLH2O Stacking gel mix (6%)(5 mL)3.4mLH2O 0.63mL 30% acrylamide:bisacrylamide (29:1) mix

Buffers for SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

20 mM HEPES500 mM NaCl 2 mM EDTA1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer C)IP7 reaction buffer(10X)250 mM HEPES,pH 7.4500 mM NaCl60 mM MgCl210 mM DTT (1 M stock was made separately, aliquoted into 100 μL and stored at -20oC).10X buffer was made and stored at 4oC. An appropriate amount was added to the reaction mix to get a final concentration of 1X.DTT was added fresh to the reaction buffer just before use

Buffer C

2 mM EDTA5 mM DTT1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer B)

20 mM HEPES pH 6.8100 mM NaCl

Buffer B

20 mM HEPES pH 6.8100 mM NaCl2 mM EDTA5 mM DTTYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer A)

Buffer A

Buffers for protein purification and IP7reaction

Buffer A1 mM EDTAin HPLC grade water (Fisher Scientific)Buffer B 1 mM EDTA(NH4)2HPO41.3 M, pH 3.8171.6 g of (NH4)2HPO4was dissolved in 750 mL of HPLC grade water. pH was adjusted to 3.8 with 75 mL of H3PO4by continuous stirring and the volume was made upto 1000 mL.Both buffers were filtered througha0.22 μm filter (Millipore) using vacuume filter apparatus (Tarsons) and degassing was performed atleast for 20 min using a vacuume pump

Buffers for IP7 purification

Wash buffer II10 mM Tris-HCI,pH 8.01 mM EDTA250 mM LiCl0.75% NP-400.75% sodium deoxycholateProtease inhibitor cocktailElutionbuffer II50 mM Tris-HCl,pH 8.0 10 mM EDTA 1% SDS

Lysis buffer50 mM HEPES,pH 7.5140 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail (added fresh)Wash buffer I50 mM HEPES,pH 7.5500 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail

Buffers for chromatin immunoprecipitation

Volume was adjusted with water to 1 L and solution was sterilized by autoclaving.Pre-hybridization/hybridization buffer (Modified Church and Gilbert buffer)0.5 M phosphate buffer (134g of Na2HPO4.7H2O,4 mL of 85%H3PO4), pH7.27% (w/v) SDS10 mM EDTA Volume was adjusted to 1 L with DEPC treated sterile water. Buffer was aliquoted into 50 mL RNase free conical tubes (Corning) and stored in -20oC.Post hybridization wash buffersWash buffer 1 2X SSC 0.1% SDS Wash buffer 2 1X SSC 0.1% SDSWash buffer 3 0.5X SSC0.1% SDSBuffers were prepared with sterile DEPC treated water

TMN buffer10mM Tris-HCl, pH 7.45 mM MgCl2100 mM NaCl Permeabilization buffer950 μLof coldwater50 μLof 10% (wt/vol)sodium N-lauroyl sarcosineTranscription assay buffer(100 μL)50mM Tris-HCl, pH 7.4100mMKCl5mM MgCl21mM MnCl22 mM dithiothreitol 0.5mM ATP 0.25 mM GTP0.25mM CTP10mM phosphocreatine2.4 units creatine phosphokinase100μCi [α-32P] UTP (3,000Ci/mmol)Alkaline denaturing solution for DNA for membrane preparation0.5 M NaCl 0.25 M NaOH Volume was adjusted to 20 mLwith sterile water. Saline Sodium Citrate (SSC) buffer (20X) 3.0 M Sodium chloride 0.3 M Sodium citrate

Buffers fortranscription run on analysis

50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC37% sucrose solution

100mM NaCl30mM MgCl250μg/mLcycloheximide 200μg/mL heparin All the components were made in DEPC treated water.Gradient buffer10% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC10% sucrose solutionTo analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.30% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC30% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.50% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC50% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.37% sucrose gradient buffer

Lysis buffer10mM Tris, pH7.4

Buffers for ribosome and polysome analysis

30%GlycerolMade in 100 mL.RNA sample loading buffer (10X)50% glycerol10mM EDTA 0.025% Bromophenol blue 0.025% Xylene cyanolInoue transformation buffer, pH 6.7(125 mL, prepared just before use)10 mM PIPES 15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O (1.361 g is dissolved in 10 mL of water separately)PIPES(0.307 g), CaCl2.2H2O (0.275 g) and KCl (2.325 g)were added to 80 mL ofsterile water while mixing with a magnetic stirrer and the pH was adjusted to 6.8with 1 N KOH. After attaining the appropriate pH, MnCl2solution wasadded slowly in aliquotes of 300 μL over 10 min,while stirring to avoidabrown precipitate.MOPS buffer(10X)0.2 M MOPS, pH 7.220 mM CH3COONa10 mM EDTABuffer was made in DEPC treated waterYeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/mLSalmon sperm carrier DNA Dimethyl sulfoxide (DMSO) Zymolyase cocktail buffer for yeast colony PCR 2.5 mg/mLZymolyase (ZymoResearch)1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

Yeast lysis buffer for genomic DNA extraction50 mM Tris-HCl,pH 8.010 mM EDTA 150 mM NaCl 1% Triton-X 1% SDSAE buffer for RNA extraction50 mMSodium acetate,pH 5.31 mMEDTA,pH 8.0Solution was made in DEPC treated water. 0.2%diethyl pyrocarbonate (DEPC)was added to the water and stirred for 12 h. To remove DEPC,water was autoclaved twice. DNA sample loading buffer (6X)15.25 mg Bromophenol blue15.25 mg Xylene cyanol

Buffers for extraction and analysis of genomic DNA and RNA

EDTA (pH 8.0)186.1 g of EDTA.2H2O was dissolved into 800 mL of water stirredvigorously and the pH was adjusted with NaOH pellets. When the pH of the solution reached8.0 EDTA dissolvedcompletely and was made upto 1000 mL with water.Tris-HCl buffer (1M)121.1 g of Tris base was dissolved in 800 mLof water and pH was adjusted to 7.2 using concentrated HCl Tris-EDTA (TE) buffer 10 mM Tris-HCl, pH 8.01 mM EDTA Tris-Acetic acid EDTA (TAE) buffer 40 mM Tris base 1mMEDTApH was adjusted to 8.4with glacial acetic acid. TAE buffer was prepared as a 50X stock solution and used at 1Xconcentration.Tris-Saline20 mM Tris-HCl, pH 7.20.9% NaCl

PhosphateBuffered Saline (PBS) 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 using HCl and NaOH beforeautoclaving. PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving

Common buffers

Buffers and solutions

Liquid scintillation cocktail5 g PPO (2,5-diphenyloxazol)0.3 g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene Volume was adjusted to 1L with toluene.MUG (4-methylumbelliferyl β-d-glucuronide)extraction buffer1 mM MUG substrate50 mM Sodium dihydrogen phosphate (pH-7.0)10 mM EDTA0.1% Triton X-1000.1% Sodium lauryl sarcosine10 mM β-MercaptoethanolLactophenol solution (100 g)25 g Lactic acid (20.66 ml)25 g Phenol 50 g Glycerol (39.77 ml)These three components were mixed together and 1 volume of lactophenol was added to 2 volumes of ethanol

CAS solutiona) 0.06 g Chrome Azurol S dye in 50 mlb) Fe (III) solution: 10 ml1 mM FeCl310 mM HClc) 0.072 g HDTMA in 40 mlAll the above three solutions were mixed together and autoclaved prior to use

Other solutions

Stripping buffer100 mM β-mercaptoethanol2 % SDS62.5 mM Tris-HCl (pH 6.7)Final volume was madeto 250 ml with water

Transfer buffer (10 X stock solution)0.25 M Tris-HCl (pH8.0)1.92 M Glycine1% SDSThe stock solution was prepared asa10 X concentrate and was diluted to 1 X concentration prior to use.1X Transfer buffer (1 litre)200 ml Methanol100 ml 10X Transfer buffer700 ml WaterTris-Buffered Saline (TBS)50 mM Tris150 mM NaClFinal pHof the bufferwas adjusted to 7.4 with HCl.Blocking buffer5% Fat-free milk0.1% Tween-20Final volume was made to 100 ml with 1 X TBS.Wash buffer (TBS-T)TBS (1 X final concentration)0.1% Tween-20Final volume was prepared with water

Buffers for western blot experiment

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

Total cell lysis buffer (Homogenization buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM Sodium fluoride*1 mM Sodium orthovanadate*1 X protease inhibitor cocktail (Sigma, P 8215)** Were added fresh before use.SDS-PAGE30% acrylamide solution29 g Acrylamide1 g N,N’-MethylenebisacrylamideDissolved in 100 ml H2O10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol blue

Buffers for protein extraction and separation by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

Phenol solution saturated with 0.1 M citrate buffer (pH 4.3 ± 0.2)was procured from Sigma (P4682)

Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSPhenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol (pH 8.0)24 ml Chloroform1 ml Isoamyl alcoholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollStock solution of the loading buffer was prepared in water as a 6 X concentrate and was added to the sample DNA to the final concentration of 1 X.RNA isolation bufferAE buffer3 M sodium acetate0.5 M EDTA (pH 8.0)Reagents used for RNA isolation were prepared in DEPC-treatedwater and stored at 4°C. For preparationof DEPC-treated water,0.1 ml DEPC was added to 100 ml waterand kept overnight onamagnetic stirrer. Followingincubation,the solution was autoclaved to remove any traces of DEPC.Acid phenol solution

Genomic DNA isolation buffersBuffer A50 mM Tris-HCl10 mM EDTA150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol (Added freshbefore use)

Buffers used for nucleicacid extraction

10 mg/ml carrier DNA 5 μlAbove-mentioned reagents were added to prepare thetransformation mixture, and the volumes indicated wereused per transformation.500-1,000 ng of desired transforming DNA was added to this transformation mixture and final volume was adjusted to 360 μl with sterile water.Carrier DNA (Sonicated salmon sperm DNA, Stratagene, 201190) washeat denatured at 95⁰C for 10 min and transferred on ice before additionto the transformation mixture.43 μl DMSO was added to each transformation mixture before heat shock.Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml zymolyase (MP Biomedicals, 0832092)1.2 M SorbitolThe cocktail was prepared in sterile water

Tris-acetic acid EDTA (TAE) buffer40 mM Tris Base0.5 M EDTAFinal pHof the bufferwas adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared as a 50 Xconcentrate and diluted to 0.5X concentration prior to use as agarose gel electrophoresis running buffer and to cast agarose gels.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESFinal pHof the bufferwas adjusted to 7.5 with NaOH.HEPES was used as a buffering agent forpreparationof different pHmedium. Buffer was filter-sterilizedby usinga0.22 μm membrane filterand stored at 4°C.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OFor preparation ofINOUE transformationbuffer,above-mentioned solutes were dissolved in appropriate amount in 800 ml of water and then 20 ml of 0.5 M PIPES(piperazine-1,2-bis[2-ethanesulphonic acid])(pH 6.7) was added. Final volume was adjusted to 1 litre with water, buffer was filter sterilized by usinga0.22 μm membrane filter and stored at -20°C. Stock solution of PIPES was preparedseparatelyby dissolving 15.1 gm of PIPES in 80 ml of water, pH was adjusted to 6.7 using 5 M KOH and volume was adjusted to 100 ml.Yeast transformation reagents1 M lithium acetate 36 μl50 % polyethylene glycol 240 μl

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4Final pH of the buffer was adjusted to 7.3with 11.6 N HCland volume was adjusted to 1 Lbefore autoclaving.PBS was prepared as a 10X stock solution and diluted to 1 X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BaseFinal pHof the bufferwas adjusted to 7.6 using 11.6 NHCl.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATE buffer was prepared as a 10 X concentrate and diluted to 1 X concentration before use

Common buffers

Buffers and solutions

3.4 mL H200.63 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)0.83 mL 1 M Tris (pH 6.8)0.05 mL 10% SDS0.05 mL 10% ammonium persulfate (APS)0.005 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)Resolving gel solution (12%)10 mL3.3 mL H204 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)2.5 mL 1.5 M Tris (pH 8.8)0.1 mL 10% SDS0.1 mL 10% ammonium persulfate0.004 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)SDS-Running buffer(Tris/Glycine/SDS)25 mM Tris-Cl192 mM glycine0.1% SDSTransfer Buffer25mM Tris-Cl190 mM glycine20% MethanolMTT dyeMTT dye was dissolved in PBS at 5 mg/mL concentration. Filtered through 0.45 μm syringe filters, and stored in dark at 4ºC

Hypotonic lysis buffer10mMTris Cl (pH 7.4)2.5mM MgCl2, 1mM PMSF 0.5% NP-40Hypotonic bufferPrewarmed 0.075 M KCl Fixatives used in this study100% ethanol kept overnight at -20ºCfor immunofluorescence by prelysis protocol70% ethanol kept overnight at -20ºCused for PI based cell cycle analysisMethanol: glacialactetic acid (3:1) for cytogenetic analysis4% Paraformaldehyde-4 g of paraformaldehyde dissolved in 100 mL waterPI staining solutionPBS containing, 0.1% Triton X-1000.2 mg RNase 20μg propidium iodideReagents required for Tris-Glycine SDS-PAGESDS-PAGE 30% Acrylamide solution29 g of acrylamide and 1 g bi-acrylamide (29:1 ratio) dissolved in 100 mL water10% SDS10 g SDS dissolved and 100 mL waterLaemmli buffer40% Glycerol 240 mM Tris/HCl pH 6.8 8% SDS 0.04% bromophenol blue 5% beta-mercaptoethanolStacking gel solution (5%) 5mL

Buffers