MOLECULAR BIOLOGY: A PROJECT APPROACH
CONTENTS I INTRODUCTION I LABORATORY ETIQUETTE AND SAFETY II GOOD LABORATORY PRACTICES III MAINTENANCE OF DISTILLATION APPARATUS IV WASHING OF GLASSWARE V DRY STERILIZATION VI HANDLING OF PIPETTE VII MOLECULAR, EMPIRICAL AND FORMULA WEIGHT VIII PLANNING A SOLUTION OF A PARTICULAR MOLARITY IX MAKING SOLUTIONS FROM HYDRATED COMPOUNDS X MEASUREMENT OF LIQUIDS WITH NORMALITY / MOLARITY XI NUMBERS FOUND ON CHEMICAL BOTTLES XII ACCURACY AND CALIBRATION XIII BUFFERS II INSTRUMENTS: PRINCIPLE AND PRECAUTIONS I WEIGHING BALANCE II SPECTROPHOTOMETER III LAMINAR FLOW IV AUTOCLAVE V CENTRIFUGE VI PH METER VII INCUBATOR VIII HOT AIR OVEN IX COMPOUND MICROSCOPE III EXPERIMENTS PART 1 MICROBIOLOGY 1 1 MICROMETRY AND MEASUREMENT OF MICROORGANISMS 1 1 1 CALIBRATION OF MICROSCOPE BY USING OCULAR AND STAGE MICROMETER 1 1 2 MEASUREMENT OF SIZE OF MICROORGANISM / SPORE 1 2 PREPARATION OF CULTURE MEDIA 1 2 1 LIQUID CULTURE MEDIUM (BROTH) 1 2 2 SOLID MEDIUM 1 2 3 AGAR PLATES AND AGAR SLANTS 1 2 4 SELECTIVE AND DIFFERENTIAL MEDIA 1 2 4 1 EOSIN METHYLENE BLUE (EMB) METHOD 1 2 4 2 MANNITOL SALT AGAR 1 2 4 3 PIKOVSKAYA AGAR 1 3 ASEPTIC METHODS 1 3 1 PHYSICAL METHODS OF STERILIZATION 1 3 2 CHEMICAL METHODS OF STERILIZATION 1 3 3 GASEOUS STERILIZATION 1 4 CULTIVATION TECHNIQUES 1 4 1 STREAK PLATE METHOD 1 4 2 POUR PLATE TECHNIQUE 1 4 3 SPREAD PLATE TECHNIQUE 1 4 4 SERIAL DILUTION TECHNIQUE 1 5 PRESERVATION AND MAINTENANCE OF CULTURES 1 5 1 MAINTENANCE BY SUB-CULTURING 1 5 2 TO PREPARE AGAR SLANTS WITH MINERAL OIL 1 5 3 STORAGE IN SOIL 1 5 4 TO STORE THE CULTURES IN LIQUID NITROGEN 1 5 5 TO STORE THE CULTURES AT -70°C 1 5 6 TO PRESERVE THE CULTURES BY LYOPHILIZATION (FREEZE DRYING) 1 6 STAINING TECHNIQUES 1 6 1 PREPARATION OF BACTERIAL SMEAR AND FIXATION 1 6 2 SIMPLE STAINING OF BACTERIA 1 6 3 NEGATIVE STAINING OF BACTERIA 1 6 4 GRAM’S STAINING OF BACTERIA 1 6 5 ACID FAST STAINING 1 6 6 ENDOSPORE STAINING 1 6 6 1 BY MALACHITE GREEN 1 6 6 2 DORNER’S METHOD 1 6 7 CAPSULE STAINING TO DETECT THE CAPSULE OR SLIME LAYER IN BACTERIA 1 6 8 FLAGELLA STAINING 1 6 8 1 LEE’S METHOD 1 6 8 2 LEIFSON’S METHOD 1 6 9 TO TEST VIABILITY OF BACTERIA BY STAINING 1 6 10 STAINING OF FUNGI 1 6 11 STAINING OF ARBUSCULAR MYCORRHIZA (AM) 1 7 ISOLATION AND ENEUMERATION OF MICROBES 1 7 1 ISOLATION AND ENUMERATION OF FUNGI FROM SOIL 1 7 1 1 ISOLATION OF FUNGI BY DILUTION PLATE METHOD 1 7 1 2 ISOLATION OF FUNGI BY WARCUP METHOD 1 7 2 ISOLATION OF SEED MICROFLORA 1 7 2 1 AGAR PLATE METHOD 1 7 2 2 BLOTTER METHOD 1 7 3 ISOLATION OF AEROMYCOFLORA 1 7 4 ISOLATION OF VESICULAR ARBUSCULAR MYCORRHAZIA (VAM) SPORES FROM THE SOIL 1 7 4 1 WET- SIEVING METHOD 1 7 4 2 FLOATATION METHOD 1 7 5 ISOLATION AND ENUMERATION OF MICROORGANISMS FROM RHIZOSPHERE 1 7 6 ISOLATION OF MICROORGANISMS FROM RHIZOPLANE 1 7 7 ISOLATION OF PROTOZOA FROM SOIL 1 7 8 ISOLATION OF CYANOBACTERIA 1 7 9 ISOLATION OF AQUATIC FUNGI BY BAITING METHOD 1 7 10 ISOLATION OF YEAST 1 7 11 ISOLATION OF RHIZOBIA FROM THE ROOT NODULES 1 7 12 ISOLATION AND CULTIVATION OF ANAEROBIC BACTERIA 1 7 12 1 CANDLE JAR METHOD 1 7 12 2 GASPAK ANAEROBIC JAR METHOD 1 7 13 ANALYSIS AND ENUMERATION OF BACTERIA IN MILK 1 7 14 PRESUMPTIVE TEST FOR COLIFORMS TO CHECK THE QUALITY OF MILK 1 7 15 MICROBIOLOGICAL EXAMINATION OF CHEESE 1 7 16 MICROBIOLOGICAL EXAMINATION OF BUTTER 1 8 COUNTING OF CELLS/ SPORES 1 8 1 MEASUREMENT OF CELLS/ SPORES BY COUNTING CHAMBER 1 8 2 COUNTING OF CELLS BY SERIAL DILUTION TECHNIQUE 1 8 3 COUNTING OF BACTERIAL POPULATION SPECTROPHOTOMETRICALLY 1 9 MICROBIAL GROWTH 1 9 1 DETERMINATION OF BACTERIAL GROWTH BY SPECTROPHOTOMETRIC METHOD 1 9 2 MEASUREMENT OF FUNGAL GROWTH BY COLONY DIAMETER 1 9 3 MEASUREMENT OF FUNGAL GROWTH BY DRY WEIGHT OF MYCELIUM 1 9 4 ESTIMATION OF BIOMASS 1 9 4 1 DRY CELL WEIGHT ESTIMATION 1 9 4 2 PACKED CELL VOLUME DETERMINATION 1 9 5 EFFECT OF TEMPERATURE ON GROWTH 1 9 6 EFFECT OF PH ON GROWTH 1 9 7 DETERMINATION OF ANTIBIOTIC SENSITIVITY BY DISC METHOD 1 10 FERMENTATION TECHNOLOGY 1 10 1 ESTIMATION OF LACTIC ACID 1 10 2 ESTIMATION OF CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER 1 10 3 BIOCONVERSION OF TANNIC ACID TO GALLIC ACID 1 10 4 DETERMINATION OF ALCOHOL PRODUCTION IN THE FERMENTED BROTH 1 10 5 ALPHA AMYLASE BIOSYNTHESIS AND MEASUREMENT OF ITS ACTIVITY 1 11 MUSHROOM CULTIVATION 1 11 1 PRODUCTION OF SPAWN FOR WHITE BUTTON MUSHROOM (AGARICUS BISPORUS) 1 11 2 CULTIVATION OF WHITE BUTTON MUSHROOM PART 2 BIOCHEMISTRY 2 1 CARBOHYDRATES 2 1 1 QUALITATIVE TEST FOR CARBOHYDRATES 2 1 1 1 MOLISCH’S TEST 2 1 1 2 IODINE TEST 2 1 1 3 BARFOED’S TEST 2 1 1 4 SELIWANOFF’S TEST 2 1 1 5 FEHLING’S TEST 2 1 1 6 BENEDICT’S TEST 2 1 1 7 BIAL’S TEST 2 1 1 8 OSAZONE TEST 2 1 1 9 TEST FOR NON REDUCING SUGARS 2 1 2 QUANTITATIVE TEST FOR CARBOHYDRATES 2 1 2 1 DETERMINATION OF TOTAL CARBOHYDRATE BY ANTHRONE REAGENT 2 1 2 2 DETERMINATION OF REDUCING SUGAR BY NELSON- SOMOGYI METHOD 2 1 2 3 DETERMINATION OF CELLULOSE 2 2 AMINO ACIDS AND PROTEINS 2 2 1 COLOR REACTION OF PROTEINS AND AMINO ACIDS 2 2 2 TITRATION CURVE OF AMINO ACIDS 2 2 3 PROTEIN ASSAY METHODS 2 2 3 1 THE ? MAX FOR PROTEINS AND AMINO ACIDS 2 2 3 2 DETERMINATION OF MOLAR ABSORBANCE COEFFICIENT (?) OF L-TYROSINE 2 2 3 3 LOWRY’S METHOD OF PROTEIN ASSAY 2 2 3 4 BRADFORD PROTEIN ASSAY 2 2 3 5 BICINCHONINIC ACID PROTEIN ASSAY 2 2 3 6 BIURET ASSAY 2 2 4 ISOLATION OF CASEIN FROM MILK 2 2 5 AMMONIUM SULPHATE FRACTIONATION OF PROTEINS 2 3 ENZYMES 2 3 1 EXTRACTION AND PURIFICATION OF ENZYMES: GENERAL STRATEGIES 2 3 2 EXPRESSION OF ENZYME ACTIVITY 2 3 2 1 TO STUDY THE EFFECT OF PH AND TEMPERATURE ON ACTIVITY OF ACID PHOSPHATASE 2 3 2 1 1 TO DETERMINE THE ACTIVITY OF ACID PHOSPHATASE 2 3 2 1 2 TO STUDY THE EFFECT OF PH ON ACTIVITY OF ACID PHOSPHATASE 2 3 2 1 3 TO STUDY THE EFFECT OF TEMPERATURE ON ACTIVITY OF ACID PHOSPHATASE 2 3 2 2 PURIFICATION OF ACID PHOSPHATASE FROM RAW WHEAT GERM 2 3 2 3 TO DETERMINE THE KM AND VMAX VALUE OF ACID PHOSPHATASE 2 3 3 EXTRACTION AND ASSAY OF ENZYMES 2 3 3 1 MALATE DEHYDROGENASE 2 3 3 2 PEROXIDASE 2 3 3 3 POLYPHENOL OXIDASE 2 3 3 4 SALIVARY AMYLASE 2 3 3 5 PAPAIN 2 3 4 IMMOBILIZATION OF AN ENZYME 2 4 LIPIDS 2 4 1 QUALITATIVE TEST FOR LIPIDS 2 4 1 1 SOLUBILITY TEST FOR LIPIDS 2 4 1 2 GREASE SPOT TEST 2 4 1 3 ACROLEIN TEST FOR GLYCEROL 2 4 1 4 LIEBERMANN-BURCHARD TEST FOR CHOLESTEROL 2 4 2 DETERMINATION OF FREE FATTY ACIDS 2 4 3 DETERMINATION OF IODINE NUMBER 2 4 4 DETERMINATION OF SAPONIFICATION VALUE 2 4 5 ESTIMATION OF SERUM CHOLESTEROL (TOTAL) 2 5 NUCLEIC ACIDS 2 5 1 DETERMINATION OF ? MAX OF NUCLEIC ACIDS 2 5 2 DETERMINATION OF DNA BY DIPHENYLAMINE METHOD 2 5 3 DETERMINATION OF RNA BY ORCINOL METHOD 2 6 CELL ORGANELLES 2 6 1 ISOLATION OF MITOCHONDRIA 2 6 1 1 ISOLATION OF MITOCHONDRIA FROM PLANT LEAVES 2 6 1 2 ISOLATION MITOCHONDRIA FROM RAT LIVER 2 6 2 ISOLATION OF CHLOROPLAST FROM PLANT LEAVES 2 7 PLANT PIGMENTS AND PHENOLICS 2 7 1 ESTIMATION OF CHLOROPHYLL 2 7 2 ESTIMATION OF CAROTENE 2 7 3 ESTIMATION OF LYCOPENE 2 7 4 ESTIMATION OF TOTAL PHENOL BY FOLIN-CIOCALTEAU METHOD 2 8 VITAMINS 2 8 1 ESTIMATION OF THIAMINE 2 8 2 ESTIMATION OF ASCORBIC ACID IN LEMON JUICE 2 8 3 ESTIMATION OF RIBOFLAVIN 2 8 4 ESTIMATION OF VITAMIN A 2 9 BIOCHEMICAL SEPARATION TECHNIQUES 2 9 1 PAPER CHROMATOGRAPHY 2 9 2 THIN LAYER CHROMATOGRAPHY 2 9 3 GEL-FILTRATION CHROMATOGRAPHY 2 9 4 POLYACRYLAMIDE SODIUM DODESYL SULPHATE GEL ELECTROPHORESIS (SDS-PAGE) 2 9 5 SILVER STAINING 2 9 6 ISOELECTROFOCUSING PART 3 MOLECULAR BIOLOGY 3 1 ISOLATION OF NUCLEIC ACIDS 3 1 1 ISOLATION OF DNA 3 1 1 1 ISOLATION OF E COLI PLASMID DNA 3 1 1 2 ISOLATION OF E COLI GENOMIC DNA 3 1 1 3 ISOLATION OF PHAGE DNA 3 1 1 4 ISOLATION OF PLANT DNA (CTAB METHOD) 3 1 1 5 ISOLATION OF MAMMALIAN DNA 3 1 1 5 1 ISOLATION OF DNA FROM MAMMALIAN TISSUE 3 1 1 5 2 ISOLATION OF DNA FROM BLOOD CELLS 3 1 1 6 ISOLATION OF CHLOROPLAST DNA 3 1 1 7 ISOLATION OF MITOCHONDRIAL DNA 3 1 1 8 AGAROSE GEL ELECTROPHORESIS 3 1 2 ISOLATION OF RNA 3 1 2 1 ISOLATION OF TOTAL RNA 3 1 2 2 ISOLATION OF MESSENGER RNA 3 1 2 3 AGAROSE GEL ELECTROPHORESIS OF RNA 3 1 2 4 QUANTIFICATION OF DNA/RNA 3 1 2 5 DETERMINATION OF DNA QUALITY 3 2 DENATURATION AND RENATURATION OF DNA 3 2 1 DETERMINATION OF MELTING TEMPERATURE (TM VALUE) OF DNA 3 2 2 DETERMINATION OF GC/AT CONTENT OF DNA 3 3 RESTRICTION DIGESTION 3 3 1 RESTRICTION DIGESTION OF DNA 3 3 2 AGAROSE GEL ELECTROPHORESIS OF DIGESTED DNA SAMPLES 3 4 POLYMERASE CHAIN REACTION (PCR) 3 4 1 DNA AMPLIFICATION BY PCR 3 4 2 RAPD ANALYSIS OF PLANT DNA 3 4 3 PCR AMPLIFICATION OF 16S RIBOSOMAL RNA 3 5 BLOTTING TECHNIQUES AND HYBRIDIZATION 3 5 1 SOUTHERN BLOTTING AND HYBRIDIZATION 3 5 2 NORTHERN BLOTTING AND HYBRIDIZATION 3 5 3 WESTERN BLOTTING AND HYBRIDIZATION 3 6 LIGATION AND TRANSFORMATION OF DNA 3 6 1 LIGATION 3 6 2 PREPARATION OF COMPETENT E COLI CELLS 3 6 3 TRANSFORMATION OF E COLI CELLS 3 6 4 TRANSFORMATION OF PLANT CELLS USING AGROBACTERIUM TUMIFACIENS 3 6 5 ISOLATION OF PLANT PROTOPLASTS 3 7 SELECTION OF RECOMBINANT CELLS 3 7 1 SELECTION BY ANTIBIOTIC RESISTANCE 3 7 2 LAC Z’ GENE SELECTION 3 7 3 GUS ASSAY REFERENCES