Identificationof bioagent

Isolation and purification of pathogen, Fusarium udum

Production of chitinase

Amino acids utilisation test (Decarboxylase/dihydrol

Amino acids utilisation test (Decarboxylase/dihydrola

Primary antibodies

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

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

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

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

For preparation of Ultra competent cells

Inoue buffer

(f) Running buffer

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

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

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