|About the book|
?It is a complete instruction manual that imparts knowledge on principles, protocols and applications on techniques of biochemistry, immunology and biotechnology accurately in a user-friendly style.? Prof R C Kuhad Head, Department of Microbiology University of Delhi South Campus
Lab Manual is intended to be a handy reference for undergraduate and postgraduate students in life science and allied fields. The book covers fundamental exercises as well as advanced protocols, along with authentic explanation of various techniques and precautions pertaining to common errors in the laboratory.
Divided into 10 sections, Lab Manual covers:
1. Techniques of biochemistry and clinical biochemistry, cell biology enzymology
2. Latest techniques of immunology
3. Isolation and cultivation of microbial and eukaryotic viruses, methods of direct gene transfer, mutagenesis and recombinant DNA technology.
Written by authors who have over a decade of experience in teaching and research, this book will serve the needs of:
? Practitioners in medical and research labs of applied biological sciences.
? Professionals from interlinked disciplines such as human nutrition, medical instrumentation, etc.
? Students of biochemistry, microbiology, immunology, genetics and biotechnology.
|About the author|
Dr. Arti Nigam is Reader at the Department of Microbiology, Institute of Home Economics, University of Delhi. She did her post graduation in biotechnology from Jawaharlal Nehru University in 1990, and was subsequently awarded her Ph D in Microbiology in 1995 from the University of Delhi South Campus.
Dr. Nigam has more than 13 years of teaching experience at graduation and post-graduation levels. Besides being a keen researcher herself, she has also supervised many students for their post-graduation dissertations. The main research areas of her interest include industrial biotechnology and biochemistry. She has published her research work in national and international journals of repute as well as participated in various conferences, seminars and symposia over these years to keep her abreast of the latest developments in her field. Dr. Nigam is a member of Association of Microbiologists of India (AMI).
Dr. Archana Ayyagari is Reader at the Department of Microbiology, Swami Shraddhanand College, University of Delhi. She was awarded her post graduation degree in Biotechnology from M. S. University of Baroda in 1990, followed by her doctoral degree in Microbiology from University of Delhi South Campus in 1996. The research areas that fascinate her are biochemistry, instrumentation, biotechnology and the scope of these interdisciplinary and applied sciences. She has served as the Head of the Department twice in the past decade.
Dr. Ayyagari was awarded a German Academic Exchange Service fellowship (DAAD fellowship) in 1999. At Max Planck Institute of Biochemistry at Munich, she carried out her post-doctoral research on cloning and hyperexpression of synthetic human ubiquitin gene in a chemolithotroph, followed by the standardization of industrial production of the same in a pilot scale bioreactor.
She has published about eight scientific research papers out of her original research work in reputed international microbiology and biotechnology journals, three popular science articles in science magazines, seven chapters in different national and international microbiology books and about a dozen abstracts of scientific papers published in abstract books of many conferences. She has attended more than twelve national and international conferences, seminars and symposia.
She received the Young Scientist Award from the prestigious Association of Microbiologists of India (AMI) in 1997. She is a member of AMI, Indian Science Congress Association (ISCA), Indian National Science Academy (INSA) and Deutcher Akademischer Austauchdienst (DAAD).
|Table of contents|
Part I: BASIC TECHNIQUES IN BIOCHEMISTRY
EXPERIMENT NO. 1: To measure pH of tap water and distilled water using pH paper strips and a pH meter.
EXPERIMENT NO. 2: To study the concept of solutions and buffers, and to prepare a buffer of a known pH.
EXPERIMENT NO. 3: (i) To study working principles of spectrophotometry and colorimetry, and preparation of the absorbance spectrum of the given dye. (ii) To prepare the absorbance spectra of a protein sample, nucleic acid sample and co-enzyme sample in UV range.
EXPERIMENT NO. 4: To prove Beer?s law experimentally, i.e., to find out the relation between the absorbance and the concentration by preparing a standard graph of p-nitrophenol.
Part II: QUALITATIVE AND QUANTITATIVE METHODS TO STUDY BIOMOLECULES
EXPERIMENT NO. 5: To perform Molisch test for identification of sugars.
EXPERIMENT NO. 6: To perform Fehling?s test to detect reducing sugars.
EXPERIMENT NO. 7: To perform Benedict?s test for reducing sugars.
EXPERIMENT NO. 8: To perform Barfoed?s test for distinguishing reducing monosaccharides from reducing disaccharides.
EXPERIMENT NO. 9: To perform Seliwanoff?s test for detection of ketose sugars.
EXPERIMENT NO. 10: To perform osazone test for reducing sugars.
EXPERIMENT NO. 11: To perform Bial?s test for detection of pentoses.
EXPERIMENT NO. 12: To quantify total sugars in the given sample using anthrone method.
EXPERIMENT NO. 13: To estimate reducing sugars in the given sample using dinitrosalicylic acid colorimetrically.
EXPERIMENT NO. 14: Estimation of reducing sugar by the Somogyi?Nelson method.
EXPERIMENT NO. 15: To detect the presence of amino acids qualitatively by Ninhydrin test, Xanthoproteic test, Glyoxylic test, modified Millon?s test, Pauly?s test, Ehrlich test, Nitroprusside test, lead acetate test and Sakaguchi test.
EXPERIMENT NO. 16: To prepare titration curve for the given amino acids and to determine their pKa values.
EXPERIMENT NO. 17: To isolate glutamic acid from gluten of wheat flour.
EXPERIMENT NO. 18: To quantify amino acids using ninhydrin reaction.
EXPERIMENT NO. 19: To carry out qualitative tests for the presence of proteins in the given sample.
EXPERIMENT NO. 20: To estimate protein quantitatively by Biuret method.
EXPERIMENT NO. 21: To estimate protein in the given sample by Folin--Lowry?s method.
EXPERIMENT NO. 22: To determine quantitatively the proteins present in a sample by Bradford method.
EXPERIMENT NO. 23: To estimate the amount of protein using ultraviolet absorbance method.
EXPERIMENT NO. 24: To estimate the protein content of given foodstuff sample by determination of nitrogen by Microkjeldahl method.
EXPERIMENT NO. 25: To isolate casein from milk.
EXPERIMENT NO. 26: To determine the isoelectric point (pI) of the given protein (casein).
EXPERIMENT NO. 27: To carry out qualitative tests to detect lipids in the given sample.
EXPERIMENT NO. 28: To determine acid value of the given fat samples and to compare acid values of fresh samples with those of corresponding stale ones.
EXPERIMENT NO. 29: To determine the saponification value of a fat.
EXPERIMENT NO. 30: To determine iodine value for the different lipid/fat sample.
EXPERIMENT NO. 31: To test the given fats and oils for unsaturation.
EXPERIMENT NO. 32: To perform fatty acid titration.
EXPERIMENT NO. 33: To estimate vitamin A in the given sample.
EXPERIMENT NO. 34: To estimate thiamine in the given food sample.
EXPERIMENT NO. 35: To estimate riboflavin in the given food sample.
EXPERIMENT NO. 36: To estimate niacin in the given sample of foodstuff.
EXPERIMENT NO. 37: To estimate ascorbic acid by titration using 2,6 ? dichlorophenol indophenol.
EXPERIMENT NO. 38: To estimate vitamin E (tocopherol) in the given sample colorimetrically.
EXPERIMENT NO. 39: To estimate DNA quantitatively by diphenylamine method.
EXPERIMENT NO. 40: To detect and estimate nucleic acids (DNA and RNA) by ultraviolet spectrophotometry.
EXPERIMENT NO. 41: To determine the melting temperature (Tm) and %GC content of the given DNA sample experimentally from the DNA melting curve.
EXPERIMENT NO. 42: To estimate RNA quantitatively using orcinol reagent.
Part III: CHROMATOGRAPHIC TECHNIQUES
EXPERIMENT NO. 43: To separate a mixture of sugars by paper chromatography.
EXPERIMENT NO. 44: To separate a mixture of sugars (glucose and maltose) by thin layer chromatography.
EXPERIMENT NO. 45: To resolve the components of a mixture of amino acids by ascending paper chromatography of the following types: (i) single dimensional and (ii) two dimensional paper chromatography.
EXPERIMENT NO. 46: To carry out chromatographic separation of the components of the given amino acid mixture using thin layer chromatography.
EXPERIMENT NO. 47: To identify fatty acids and other lipids in the given sample by thin layer chromatography (TLC).
EXPERIMENT NO. 48: To resolve various lipid components in the given mixture by using gas liquid chromatography (GLC).
EXPERIMENT NO. 49: To carry out ammonium sulfate precipitation of amylase enzyme present in the crude protein extract.
EXPERIMENT NO. 50: To carry out dialysis for desalting ammonium sulfate precipitated amylase.
EXPERIMENT NO. 51: To carry out desalting of the given protein by column chromatography.
EXPERIMENT NO. 52: To purify the amylase present in the crude preparation following ion-exchange chromatography.
EXPERIMENT NO. 53: To separate the mixture of hemoglobin and K2Cr2O7 by gel filtration chromatography using Sephadex G-50 gel, and to estimate elution volume and percent recovery.
EXPERIMENT NO. 54: To purify messenger RNA by affinity chromatography technique.
EXPERIMENT NO. 55: To resolve the given mixture of proteins by native polyacrylamide gel electrophoresis (PAGE).
EXPERIMENT NO. 56: To determine the molecular weight of the given protein by SDS-PAGE.
EXPERIMENT NO. 57: To perform silver staining for detection of small quantities of protein electrophoresed on a polyacrylamide gel.
EXPERIMENT NO. 58: To carry out isoelectric focusing of the given protein samples.
EXPERIMENT NO. 59: To resolve the given DNA sample by agarose gel electrophoresis.
EXPERIMENT NO. 60: To determine the N-terminal amino acid in the given protein.
EXPERIMENT NO. 61: To identify the C-terminal amino acid in the given protein sample.
Part IV: CLINICAL BIOCHEMISTRY
EXPERIMENT NO. 62: To carry out routine analysis of the given urine sample for (i) physical appearance (ii) chemical tests.
EXPERIMENT NO. 63: To estimate sugar in the urine by Benedict?s quantitative method.
EXPERIMENT NO. 64: To use commercially available readymade strips for the detection of glucose in the given urine sample.
EXPERIMENT NO. 65: Qualitative test for detecting proteins present in a urine sample.
EXPERIMENT NO. 66: To test urine sample for bile salts and bile pigments.
EXPERIMENT NO. 67: To test the given urine sample for the presence of ketone bodies.
EXPERIMENT NO. 68: To test the given urine sample for the presence of blood.
EXPERIMENT NO. 69: To determine the concentration of glucose in blood based on glucose oxidase activity.
EXPERIMENT NO. 70: To perform glucose tolerant test (GTT) using blood samples of a normal and diabetic person.
EXPERIMENT NO. 71: To estimate hemoglobin content of the given blood sample using (i) Sahli?s hemoglobinometer; (ii) colorimetric method.
EXPERIMENT NO. 72: To estimate serum cholesterol of the given sample by (i) Liebermann?Burchard method, (ii) Salkowski reaction and (iii) Cholesterol oxidase activity.
EXPERIMENT NO. 73: To demonstrate the presence of cholesterol and its esters microscopically by Schultz?s method.
EXPERIMENT NO. 74: To carry out extraction and estimation of serum triglycerides.
EXPERIMENT NO. 75: To estimate total plasma proteins and to separate these proteins using cellulose acetate paper electrophoresis, and by precipitation with salt.
EXPERIMENT NO. 76: To carry out cellulose acetate paper electrophoresis for hemoglobins.
EXPERIMENT NO. 77: To perform liver function test (LFT).
Part V: ENZYMOLOGY AND CELL BIOLOGY
EXPERIMENT NO. 78: Estimation of amylase enzyme activity (I.U.) and calculation of specific enzyme activity.
EXPERIMENT NO. 79: To determine kinetic properties of amylase.
EXPERIMENT NO. 80: To estimate acid phosphatase activity and to obtain the progress curve of the enzyme.
EXPERIMENT NO. 81: Estimation of alkaline phosphatase activity present in serum.
EXPERIMENT NO. 82: To estimate the activity of glucose ? 6 ? phosphatase.
EXPERIMENT NO. 83: To estimate the levels of cellulases in the given sample.
EXPERIMENT NO. 84: To estimate lipase activity using germinating oilseeds as the enzyme source.
EXPERIMENT NO. 85: To estimate alkaline protease in the given enzyme sample.
EXPERIMENT NO. 86: To monitor catalase activity in the given aerobic tissue sample.
EXPERIMENT NO. 87: To estimate urease activity in germinating moong seeds.
EXPERIMENT NO. 88: To estimate succinate dehydrogenase in germinating moong seeds.
EXPERIMENT NO. 89: To estimate invertase using commercially available yeast tablets as the enzyme source.
EXPERIMENT NO. 90: To detect the glucose isomerase present intracellularly in Bacillus sp.
EXPERIMENT NO. 91: To estimate glucose-oxidase activity in the given sample.
EXPERIMENT NO. 92: To carry out screening test for glucose-6-phosphate dehydrogenase, and its estimation in the given blood sample.
EXPERIMENT NO. 93: To study typical prokaryotic and eukaryotic cells under microscope.
EXPERIMENT NO. 94: To study various stages of mitotic cell division using onion root tips.
EXPERIMENT NO. 95: To study various stages of meiosis using grasshopper testis.
EXPERIMENT NO. 96: To perform homogenization of rat liver tissue, and obtain its subcellular fractions.
EXPERIMENT NO. 97: To identify the subcellular fractions of rat liver homogenate that contains DNA and RNA.
EXPERIMENT NO. 98: To identify subcellular fractions of rat live homogenate by studying the distribution of marker enzymes in the fractions. Part VI: BASIC AND APPLIED IMMUNOLOGY
EXPERIMENT NO. 99: To determine the blood group of given blood sample by slide agglutination.
EXPERIMENT NO. 100: To determine total leucocyte count (TLC) of the given blood sample.
EXPERIMENT NO. 101: To determine the differential leucocyte count (DLC) of the given blood sample.
EXPERIMENT NO. 102: To perform single radial immunodiffusion by Mancini?s technique.
EXPERIMENT NO. 103: To perform double immunodiffusion (DID) by Ouchterlony?s method.
EXPERIMENT NO. 104: To carry out immunoelectrophoresis.
EXPERIMENT NO. 105: To estimate the antigen concentration using rocket electrophoresis.
EXPERIMENT NO. 106: To carry out Western blot analysis for a specific protein.
EXPERIMENT NO. 107: To produce polyclonal antibodies against a purified antigen.
EXPERIMENT NO. 108: To purify IgG from serum sample by using the technique of affinity chromatography and confirm the result by double immunodiffusion.
EXPERIMENT NO. 109: To link the enzyme horse radish peroxidase with anti-BSA.
EXPERIMENT NO. 110: To perform enzyme-linked immunosorbent assay (ELISA) by (i) direct, (ii) indirect, and (iii) sandwich methods.
EXPERIMENT NO. 111: To perform dot immunobinding assay to detect antibodies present in the serum.
EXPERIMENT NO. 112: To carry out HIV-DOT for rapid screening of HIV-I and HIV ? II antibodies in serum.
EXPERIMENT NO. 113: To detect pregnancy using commercially available kits.
EXPERIMENT NO. 114: To study complement fixation and to calculate the antibody titre in the given serum sample.
EXPERIMENT NO. 115: To perform Widal test for diagnosis of typhoid.
EXPERIMENT NO. 116: To carry out serological examination for diagnosis of syphilis.
EXPERIMENT NO. 117: To detect syphilis spirochaete using indirect fluorescent antibody (IFA) technique.
Part VII: ISOLATION AND CULTIVATION OF BACTERIA, VIRUSES AND PLANT PROTOPLASTS
EXPERIMENT NO. 118: Cultivation of E. coli and isolation of its plasmid DNA by miniprep method.
EXPERIMENT NO. 119: To cultivate lambda phage (?) using an appropriate E. coli culture.
EXPERIMENT NO. 120: To isolate the DNA of bacteriophage lambda.
EXPERIMENT NO. 121: To isolate Tobacco Mosaic Virus (TMV) from infected tobacco plant leaves.
EXPERIMENT NO. 122: To culture animal viruses on various membranes of a developing chick embryo.
EXPERIMENT NO. 123: To carry out isolation, purification and culture of protoplast.
Part VIII : GENE TRANSFER METHODS
EXPERIMENT NO. 124: To carry out bacterial transformation using E. coli and a plasmid (pBR 322).
EXPERIMENT NO. 125: To demonstrate bacterial conjugation.
EXPERIMENT NO. 126: To carry out bacterial transduction of E. coli with phage P1.
EXPERIMENT NO. 127: To transfect E. coli with M13 phage DNA.
Part IX: MUTAGENESIS
EXPERIMENT NO. 128: To prepare a survival curve for the given bacterial culture using germicidal ultraviolet radiation as a mutagen.
EXPERIMENT NO. 129: To prepare a master plate and carry out replica plating of the master plate.
EXPERIMENT NO. 130: To carry out Ames test for detection of a possible chemical carcinogen.
EXPERIMENT NO. 131: To isolate petite mutants of Saccharomyces cerevisiae.
Part X: RECOMBINANT DNA TECHNOLOGY
EXPERIMENT NO. 132: To induce the biosynthesis of b-galactosidase in E.coli (strains i+ and i).
EXPERIMENT NO. 133: To isolate chromosomal DNA from E. coli.
EXPERIMENT NO. 134: To isolate low copy number and high copy number plasmids from E. coli.
EXPERIMENT NO. 135: To isolate the genomic DNA of eukaryotic cells.
EXPERIMENT NO. 136: To purify the plasmid DNA obtained by: (i) column chromatography, and (ii) CsCl density gradient method.
EXPERIMENT NO. 137: To carry out restriction endonuclease digestion and agarose electrophoresis of pBR 322.
EXPERIMENT NO. 138: To carry out ligation of a foreign insert to the vector pBR 322.
EXPERIMENT NO. 139: To study the effect of alkaline phosphatase treatment on plasmid DNA on ligation efficiency.
EXPERIMENT NO. 140: To extract total RNA from E. coli.
EXPERIMENT NO. 141: To construct a partial genomic library of E. coli chromosomal DNA (sized 1.5- 2.5 kbp) using Bam HI to restrict the DNA and pUC 18 as a vector.
EXPERIMENT NO. 142: To carry out Southern Blotting.
EXPERIMENT NO. 143: To amplify DNA isolated from the given source by PCR (polymerase chain reaction).