Yashwantrao Chavan Maharashtra

Open University

Post Graduate Degree Programme (Bio-Technology)

SBT075: Lab Course M. Sc. (Bio-Technology)

Lab

Workbook

Yashwantrao Chavan Maharashtra Open University

Vice-Chancellor-Dr. Rajan Welukar

Expert Advisory Committee

Mr. Manoj Killedar Director, School of Science & Technology, Y.C.M. Open University, Nashik Mrs. Sunanda More School of Science & Technology, Y.C.M. Open University, Nashik Mrs. Chetana Kamlaskar School of Science & Technology, Y.C.M. Open University, Nashik Dr. Sunil Ganatra 135, Krushnakunj, Toata Colony, Lakadganj, Nagpur Prof. Indira Ghosh Bio-Informatics Center, University of Pune, Pune Prof. Urmila Kulkarni-Kale Bio-Informatics Center, University of Pune, Pune Prof. Dr. Piyali Kar Maharashtra Education Foundation, Foundation Towers, Sector 11/20, CBD Belapur, Navi Mumbai

Course Writer
Mr. Pravinkumar Domade G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345, Kingsway Nagpur

Course Editor
Dr. Suchitra Godbole G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345, Kingsway Nagpur

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Mrs. Sunanda More School of Science & Technology, Y.C.M. Open University, Nashik

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Yashwantrao Chavan Maharashtra Open University, Nashik Printed & Published by: Shri. S.P. Kowale, Registrar, Y.C.M. Open University, Nashik

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List of Experiments of Semester 07 Biotechnology Lab

S. No. 1 2 3 4 5 6 7 Name of the Experiments Laboratory Rules: Basic Rules of a Microbiology Laboratory Preparation of solution of given Molarity ,Molality and Normality Preparation of buffer of specified pH using pH meter Determination of isoelectric PH of Protein The detection of changes in the conformation of bovine serum albumin (BSA) by viscosity measurement and the effect of PH on the conformation of bovine serum albumin. Estimation of sugar by DNSA method Protein estimation by Lowry/Biuret method Separation of amino acid by circular paper chromatography Separation of amino acid by Paper Electrophoresis The estimation of DNA by the diphenylamine reaction a ) The estimation of DNA by the diphenylamine reaction b ) Estimation of RNA by Orcinol reagent. Fractionation of Protein salt precipitation, solvent precipitation, isoelectric precipitation a) Demonstration of starch hydrolysis by given bacterial culture b) Demonstration of amylase production by Aspergillus niger c) Demonstration of Protein (Gelatin) hydrolysis d) Demonstration of Fat hydrolysis (lipase activity) by a bacterial culture e) Demonstration of Uease Production i.e. urea Hydrolysis a) To estimate the activity of - amylase Page No.

10 11

12 13 14 15 16 17 1 1 21 22 23 24

To study the effect of pH on activity of amylase.

To study the effect of temperature on activity of enzyme ( amylase) Effect of inhibitor on enzyme activity To determine the effect of substrate concentration on enzyme activity Evaluation of KINETIC CONSTANT [Km and Vmax] of purified enzymes Separation of green plant pigments by column chromatography To calculate the mean ,median ,mode of the given problems To find out whether the dihybrid ratio is good to fit or not in the following two crosses from the data given using 2 test A bag contains 10 pink, 10 yellow, 10 orange and 20 red beads I. What is probability of drawing 1 yellow bead. II. What is probability of drawing 1 yellow and 1 red bead. Calculate standard error (sampling error) from the observation obtained by drawing samples randomly for 25 times of sizes 5 beads at a time from population for getting pink beads.

BASIC RULES OF A MICROBIOLOGY LABORATORY

A microbiology laboratory is a place for working with a variety of microorganisms. Since several culture media are prepared and organic materials are present, the chances exist for the presence of high spectrum of microbial community. Secondly, while working with pure culture one should always follow the microbiological rules so that neither the experiment should be unsuccessful nor any hazard may occur. If a large number of students are working in a microbiology laboratory, they should be aware what to do or what not? What are the apparatus/instruments/equipments present in the laboratory and what is their functioning? What are the chemical solutions and stains and how to handle? How should the students enter in the microbiology laboratory and how should they work? Therefore, the freshers such as students, teachers, laboratory assistants and helper must follow the following guidelines: 1) Always wear an apron (a white coat or gown) before entering the microbiology laboratory to protect from microbial contamination and laboratory hazards. At regular intervals get the apron washed. 2) Cut nails regularly. 3) Tie long hairs back to avoid contamination and fire hazard. 4) Keep your working laboratory bench clean of everything. Nothing should be laying on the bench. 5) Never keep books, purses, bags, etc. on the working bench. 6) Always wash your hands with soap in running tap water before and after the work. 7) Clean your working bench with ethanol (70%) or phenol (1:100). 8) Never spit and smoke in the laboratory. 9) Dont put anything of the laboratory(e.g. pencil, thread, labels, inoculation needle, pins, etc) in your mouth, ears, nose and eyes. 10) Dont put your fingers in your eyes, ears, mouth. It may facilitate the chance of infection by pathogenic microorganisms. 11) Dont eat or drink or talk while working with microorganisms. 12) Dont mishandle the chemical solution, stains, spirit lamp, UV light, instruments/apparatus or electricity. 13) Always keep the burner at distance from the organic solvents. Your sincere care will avoid fire accident. The burner must be turned off soon after the use. 14) Always maintain aseptic condition while working with microorganisms. 15) Always use flame sterilised inoculation needle/loop. 16) Dont open the culture tubes/plates directly and never inhale them nor observe with naked eyes. 17) Open the culture tubes/plates near the vicinity of flame of the burner. 18) While working with broth culture dont suck the suspension with mouth. Always use pipette sucker. 19) After completion of work always label the cultures with names, code and date of work. It will help recording the data. 20) Always keep plates in tiers and culture tubes in upright in basket or racks. Finally, transfer all the culture in the incubator at desired temperature of where ever to keep. 21) Never leave your cultures on working table or seat. 22) Clean the working table/bench when the work is completed. 23) Clean lenses of objective with tissue paper. 24) Keep the stains, reagents, stock cultures to their respective places when the work is completed. 25) After completion of work keep your slides/pipette/culture tubes/plates in container and steam sterilize before washing. 26) Record your result at time. 27) For any difficulty, ask your laboratory assistant or concerned teachers.

Experiment No: 1
Aim : Preparation of solution of given Molarity, Molality and Normality. Theory and Principle: Normality : The number of gram equivalent of the solute present per liter of its solution . N = Weight in gram of solute/ litre of solution Equivalent weight in gram. Or = equivalent weight/ 1000. Molarity : gram molecular weight of solute per litre of solution . M = Molecular weight of solute 1000ml of solvent . Molality : It represent the number of moles of solute present in 1000g of solvent . 1m = Molecular weight of compound 1000g of solvent . Procedure : 1. Weigh the required amount of solute. 2. Dissolve in requived amount of solvent . A. Prepare 0.1N, 1M, 0.2M Solution of NaOH. Molecular weight of NaOH = 23+16+1 = 40 Equivalent weight of NaOH = molecular wt. No.of replacable H/OH = 40 a. To prepare 0.1 N solution of NaoH . To prepare 1N solution: Dissolve 40g NaOH in 1000 ml of solvent . 0.1N NaOH 4g of NaOH in 1000 ml of solvent (Distilled Water) Molarity = Molecular weight / 1000ml of solvent . For 1M of NaoH : Dissolve 40g NaoH in 1000ml of solvent (D.W.) Molality = molecular weight of compound 100g of solvent For water; volume = mass/ density = 1000ml = mass of water 1g/ml =1000g = mass of water For 1M of NaoH = 40g of NaoH dissolved in 1000ml of water There fore, 0.2M Of NaoH= 8g of NaoH dissolved in 1000ml of water B. To prepare 0.5N, 2M, 0.4M of HCL in 1000ml of solvent Molecular Weight of Hcl = 1+35.5=36.5 For Hcl specific gravity = 1.18 Volume = Mass/specific gravity =36/1.18

Therefore for 1N Hcl For 0.5N Hcl For 1M Hcl For 2M Hcl Result:

= 30.51ml = Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.) = 15.25ml Of Hcl in 1000ml of solvent (D.W.) =Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.) =Dissolve 61.02ml of Hcl in 1000ml of Solvent(D.W.)

Experiment No: 2
Aim :- Preparation of buffer of specified pH using pH meter. Theory:- Buffers are defined as substances that resist changes in the pH of the system. Weak acids or bases, in the presence of their salts with strong bases or strong acids respectively form buffer system. Ex: Phosphate/monohydrogen phosphate. Carbonic acid/bicarbonate Proteins/proteinate. pH:- The pH of the solution is the value with which defines its hydrogen ion concentration in aqueous solution, it is relative strength of hydrogen ion reach species called acid and the hydrogen ion deficient species called base which determines the net pH of a solution. Measurement of pH :pH indicators:- These are usually organic compounds of natural or synthetic origin whose colour is dependent on the pH of the solution. Indicators are dependent on the pH of the solution. Indicators are usually weak acids which dissociates in solution. pH meter:- The most reliable and accurate method for the routine measurement of pH is the pH meter in which a change in pH is measured as change in electrical potential. If a metal rod is placed in solution of its salt, it acquires potential. If two dissimilar metals are dipped into the solution of their salts, the difference in potential can be measured or calculated from the two separate potentials. the standard electrode is thus required against which the potential of other electrodes can be compared. this is the standard hydrogen electrode , consisting of platinum rod dipped in the aqueous solution with a given H+ activity in which hydrogen gas is bubbled continuously at 1 atmospheric pressure. But this is too cumbersome to be used, as reference electrode for routine use, other secondary reference electrode of known potential in relation to standard hydrogen electrode are used. Example:- Calomel electrode , glass electrode. Precautions :1.The glass electrode is fragile and must be handled with care. 2.Electrode must not be left to dry. 3.The temperature compensation dial must be set before it is calibrated as potential is produced dependent on temperature. 4.The meter must be calibrated first with a standard buffer of pH. 7 and then with pH. 4 or pH. 9. Procedure :1. Prepare the solution of given molarity as per the table. 2. Do the addition for specific buffer as per the table. 3. Make up the volume up to 1000ml. 4. Check the pH with the help of pH meter. Observation Table:S.NO. 1. 2. 3. 4. 5. pH 2 4 6 9 10 SOLUTION TO BE USED 0.2M KCl + 0.2M HCl 0.2M succinic acid + 0.2M NaOH 0.2M succinic acid + 0.2M NaOH 0.1M KCl + 0.1M H3BO3 1M NaHCO3 + 1M Na2CO3 VOLUME IN ml 65ml + 250ml 250ml + 100ml 250ml + 435ML 250ml+250ml+208ml 13.8ml + 12ml

Result:- The buffer solution of specific pH were prepared with the help of pH meter.

Experiment No: 3
Aim: Determination of isoelectric PH of Protein. Principle: The PH at which a Protein is least soluble is its isoelectric pH , defined as the pH at which the molecule has no net electric charge and fails to move in an electric field. Under these condition there is no electrostatic repulsion between neighbouring protein molecules, and they tend to coalesce and precipitate. However, at pH values above the isoelectric point all the protein molecules have a net charge of the same sign. They therefore repel each other, preventing coalescence of single molecules into insoluble aggregates. Some protein are virtually insoluble at their isoelectric pH. Since different proteins have different isoelectic pH values, because their content of amino acids with ionizable R groups differs, they can often be separated from each other by isoelectric precipitation. Materials and reagents 1) 0.005N HCl : take 83 ul of HCl and make up the volume to 200 ml with distilled water. 2) Skimmed Milk or Casein : suspend 10 gm of Skimmed Milk in 200 ml distilled water 3) Albumin Solution : Dissolve egg white from 2 egg in 200 ml distilled water. Procedure: Carry out the experiment as per the protocols given below in the table-1 and table-2 Table-1 Test tubes 1 2 3 4 5 6 Reagents Skimmed 15 15 15 15 15 15 Milk 0.005N 1 2 3 4 5 6 HCl Stand for 30 minutes and determine PH of each tube Table-2 Test tubes 1 2 3 4 5 6 7 Reagents Albumin 15 15 15 15 15 15 15 0.005N 1 2 3 4 5 6 7 HCl Keep the tubes in water bath at 50oC and determine the PH 8 15 8 9 15 9 10 15 10 7 15 7 8 15 8 9 15 9 10 15 10

Result : The isoelectric PH of the given protein solution has been found to be---------

Experiment No: 4
Aim: The detection of changes in the conformation of bovine serum albumin (BSA) by viscosity measurement and the effect of PH on the conformation of bovine serum albumin. Principle: high concentration of urea causes unfolding ie denaturation of proteins by weakening the hydrophobic bonds that maintain the tertiary structure. This change in the protein conformation leads to a less compact molecule with a larger viscosity than the native protein. Such changes in the tertiary structure can be readily followed using an Ostwald viscometer (see figure),. This , essentially consist of a capillary tube down which a known volume of protein solution is allowed to flow under gravity. The time taken for this flow is measured (t1) and also that of the solvent (t0); the relative viscosity is then given by. rel = 1/0 = (t1/t2) x (1/0) where 1 is the viscosity of the protein solution of density 1 and 0 the viscosity of the solvent of density 0. If the densities are taken to be the same then the expression simplifies to rel = t1 / t0 Einstein has shown that, for spherical molecules, the relative viscosity is related to the concentration of the molecule ( c ) and the partial specific volume (V ), which is the volume occupied by the molecule and its bound water: rel = 1+ 2.5cV Materials Viscosity is very sensitive to temperature, so all solutions an the viscometer must be kept at 30oC in the water bath. 1. Ostwald viscometer 2. water bath at 30oC 3. Potssium Chloride (100mmol/liter) 4. Urea solutions (0.5,1,2,34,6, and 8 mol/liter in 100 mmol/liter KCl) 5. Bovine Serum Albumin (10g/liter in 100mmol/ liter KCl and the above urea solutions) 6. stop watch accurate to at least 0.1s Method Always use the viscometer by one limb only and never squeeze the two arms together. Rinse the viscometer with KCl solution and place it in position in water bath by carefully clamping one limb. Check that it is vertically using the plumbline and introduce exactly 20 ml ( or the volume marked on the viscometer) of KCl solution at 30oC in the bulb A with a syringe or Pipette. Leave for 5 minute to equilibrate, then either apply positive pressure to the wide limb (I) or gentle suction to the other limb (II) until the meniscus rises to upper graduation mark B. release the pressure and measure the time ( to the nearest 0.1s) for the liquid to flow between the two graduation marks B and C. Repeat the experiment until the flow time agree within 0.2s and calculate the average flow time. Repeat the whole procedure

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with the urea solution alone (t0), which are the solvents, and then with bovine serum albumin dissolved in the urea (t1). Plot the values of t0 and t1 against the concentration of urea and join up the points with smooth curves. Select convenient concentration of urea and calculate the relative viscosities (t1/t0) using the values from the curves. This ensures that any slight errors involved in the determination of t1 and t0 are not magnified on taking the ratios. Finally prepare the graphs of the relative viscosity against the concentration of urea and comment on the results. In additions , calculate the partial specific volume of serum albumin in 10 mmol/literKCl and in 8 mol/ liter urea. Assume that the molecule remains spherical so that Einsteins equation is valid. For the determination of PH on the conformation of bovine serum albumin Material required as above experiment and PH meter

Method Using the ostwald viscometer, follow the structural change in albumin dissolved in 100 mmol/KCl and distilled water as the PH is varied over the range 2-12. Comment on the results

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Experiment No: 5
Aim: Estimation of sugar by DNSA method Principle: This method tests for the presence of free carbonyl group (c=o), the so called reducing sugar. This involves the oxidation of the aldehyde functional group present;for example; in glucose and the ketone functional group in fructose. Simultaneously 3,5 dinitrosalicylic acid (DNS) is reduced to 3amino, 5 nitrosalicylic acid under alkaline conditions.

The above reaction scheme shows that one mole of sugar will react with one mole of 3,5 dinitrosalicylic acid. Different reducing sugars generally yield different colour intensities ;thus it is necessary to caliberate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar also competes for the availability of 3,5 dinitrosalicylic acid. Although this is a convenient and relatively inexpensive method, due to the relatively Low specificity, one must run blanks diligently if the colorimetric result are to be interpreted correctly and accurately. When the effects of extraneous compounds are not known one can effectively include a socalled internal standered by first fully developing the color for the unknown sample, then a known amount of sugar is added to this sample. The increase in the absorbance is equivalent to the incremental amount of sugar added. REQUIRMENTS: 1 Test tubes, Pipettes, Spectrophotometer 2 NaoH 3 DNSA 4 Standerd giucose solution PROCEDURE: 1 Add 3ml of DNSA reagent to 3ml of glucose sample in a lighly capped test tube.(To avoid the loss of liquid due to evaporation, cover it with paraffin film.) 2 Heat the mixture at 90C for 5-15min to develop the red brown colour. 3 Add 1ml of 40% potassium tartarate(Rochelle salt)solution to stabilize the colour. 4 After cooling at room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575 nm. RESULT: Concentration of unknown was found to be ___________mg/ml.

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Experiment No: 6
Aim- Protein estimation by Lowry/Biuret method. PrincipleThe principle of this method is based on the facts that the Folin-Ciocalteu regents reacts with aromatic residues of proteins and yields blue color which in turn is read in colorimeter. The different proteins contain different aromatic residues. Blue color develops because the alkaline copper reacts with proteins; tyrosin and tryptophan present in protein reduce phosphomolybdate. (present in Folin-Ciocalteu reagent) Requirements1.1N NaOH Solution. 2.ALKALINE SODIUM CARBONATE Solution-Dissolve 20gm of Na2 CO3 in 100 ml of 0.1N NaoH solution, prepare fresh. 3.COPPER SULPHATE-SODIUM POTASSIUM TARTRATE Solution- Dissolve 0.5g/lt of CuSo4.5H2o in 1% OF sod-pot. tartrate solution, prepare fresh. 4.ALKALINE COPPER REAGENT- Mix 50ml of reagent 2 and 1ml of reagent 3 only on the day of use. 5.FOLIN-CIOCALTEAU REAGENT-It is a solution of Sodium tungstate and Sodium molybdate in phosphoric and hydrochloric acids (available commercially). Dilute the commercial reagent with an equal volume of distilled water only on the day of use. Procedurea) Take a test tube, transfer 1N NaoH solution and heat up to 100o C. b) Suspend 1 ml of protein sample into the above solution for 4-5 minutes. c) Add 5ml of reagent 4, mix properly and leave this mixture at room temp for 10 minutes. d) Add 0.5 ml of Folin-Ciocaltue reagent rapidly with immediate mixing. e) Leave it for 30 minutes; thereafter measure the absorbance of solution at750 nm in the colorimeter. PROTOCOLSr no Stock of protein (ml) 1 2 3 4 5 6 7 8 9 10 11 12 13 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Unknown 1 Unknown 2 Distilled water (ml) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Concentration of protein 0 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 Alkaline Reagent 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml Fcr (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 O.D

Result- Concentration of protein in unknown sample was found to be Unknown 1 mg/ml Unknown 2 mg/ml

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Experiment No: 7
Aim: - Separation of amino acid by circular paper chromatography. Requirement: - Whattman filter paper, Capillary tube, Cotton wicks, Spray bottle. Formula: - Rf = Distance travelled by solute /Distance travelled by solvent. Principle: - Chromatography is sensitive technique used for rapid and efficient analysis and separation of components of a mixture and purification of compounds. This experiment is based on the partition chromatography principle i.e. if to a mixture of two immiscible liquids A and B, a substance which is soluble in both A and B is added, then it distributes itself in such a way that the ratio of its concentration in two liquid A and B is constant at a particular temperature. Separation of different constituents takes place because each constituent distribute itself to different degree between the solvent which flows down the column and stationary liquid. In paper chromatography paper is used as an inert support with one solvent as stationary or immobile phase. Radial paper chromatography shows radial development. This experiment is based on differential migration of individual components of a mixture through a stationary phase. The movement of mobile phase is due to capillary action and its flow is outward from a central spot. Solvent having flow of solvent varies inversely as its viscosities. If the components to be separated are colourless then their presence is detected by spraying a suitable solvent called developing reagent i.e. Ninhydrin on the chromatogram, when various components become visible, the process is known as development. The ratio of distance the substance moves compared with the distance reached by solvent both measured from the point of application of sample is known as the Rf i.e. retention factor. Procedure:1. In this technique a circular filter is employed and then various materials to be analyzed are placed at the centre. 2. After drying the spot paper is placed horizontally on petridish possessing the solvent so that the wick of paper dips into solvent. 3. Solvent rises through wick and moves sufficient distance so that components get separated. Result: - The Rf value of following amino acid was found to beGlutamic acid Tryptophan Tyrosine -

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Experiment No:
Aim : The separation of amino acid by Paper Electrophoresis Principle: the charge carried by the molecule depends on the PH of the medium and this is illustrated in the case of three amino acids: Aspartic acid, Histidine and lysine. Electrophoresis at low voltage is not usually used to separate low molecular weight compounds because of diffusion, but it is easier to illustrate the relationship between charge and PH with amino acids than with proteins or other macromolecules. At PH 7.6, Histidine will carry zero net charge, Aspartic acid will be negatively charged and lysine will be positively charged Structures

These three amino acids can therefore be readily separated by electrophoresis. Glucose, an uncharged molecule, is included in the mixture to check for any movement of the origin due to electro-osmosis. Materials 1 horizontal electrophoresis apparatus\ 2 Power packs 3 amino acids (aspartic acid, histidine, lysine and a mixture of all three in tris-aetate buffer containing 10 g/ liter glucose) 4 tris-acetate buffer(0.007 mol/liter, PH 7.6) 5 Ninhydrin location reagent (Dissolve 0.2 g I 100ml of acetone just before use ) 6 Paper strips (10 cm x 2.5 cm) 7 Aniline-diphenylamine reagent 8 Citrate buffer (0.07 mol/liter, PH 3.0) 9 Oven at 110 oC Method Fill both parts of each electrode compatment with buffer solution to the same level: check this by arranging the siphon between them. Remove the siphon, place five filter paper strips as shown, and carefully apply a streak of the amino acid mixture to two of these , avoiding the edge of the paper. Streak three other paper strips with three other paper strips with only one amino acid and

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run all five electrophoresis strips together. Wet the paper from each electrode compartment to within a few centimeters of the point of application, leave the rest to be wetted by capillary attraction,, and immediately switch on the current . this way there is minimum spreading of the sample. Carry out electrophoresis for 3h at 8 V/cm, remove the strips , and dry in an oven at 110oC. Develop one of the strips containing mixture for glucose and dip the remaining four strips rapidly in freshly prepared Ninhydrin Solution: allow the acetone to evaporate in the air and develop the colours by heating in the oven for a few minutes. Identify the amino acids and check for any electro-osmosis. Repeat the experiment with 0.07 mol/liter citrate buffer, PH 3.0 .

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Experiment No: 10 a
Aim : The estimation of DNA by the diphenylamine reaction Principle : when DNA is treated with diphenylamine reaction under acid conditions, a blue compound is formed with a sharp absorption maximum at 595 nm. This reaction is given by 2deoxypentoses in general and is not specific for DNA. In acid solution, the straight chain form of a deoxypentose is converted to the highly reactive -hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. In DNA, only the deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present. Materials 1) DNA (commercial sample) 10 mg 2) RNA (commercial sample) 10 mg 3) pig spleen DNA solution 10 mg 4) yeast RNA solution 10 mg 5) buffered saline 500 ml ( 0.15 mol/liter NaCl: 0.015 mol/liteer sodium citrate, PH 7) 1 liter 6) diphenylamine reagent. ( dissolve 10g of pure diphenylamine in 1 liter of glacial acetic acid and add 25 ml of concentrated sulphuric acid. This solution must be prepared fresh) 7) boiling water bath Method Dissolve 10 mg of nucleic acid in 50ml of buffered saline, remove 2 ml and add 4 ml of diphenylamine reagent. Heat on a boiling water bath for 10 minutes, cool and read the extinction at 595 nm. Read the test and standards against water blank. Assay the isolated nucleic acids and the commercial samples for DNA.

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Experiment No: 10 b
Aim: Estimation of RNA by Orcinol reagent. Principle: It is a general reaction for pentoses and depends on the formation of furfural. When the pentose is heated with concentrated HCl, the Orcinol reacts with furfural in the presence of ferric chloride as a catalyst, giving a green colour. Only the purine nucleotide gives significant reactions. Requirements: A. REAGENTS: 1. Orcinol reagent: Dissolve 1gm of Ferric chloride in 1litre of Concentrated HCl and add 35ml of 6 %(w/v) Orcinol in absolute alcohol.{total volume 1litre/35ml}. 2. Standard RNA: Dissolve 10mg of RNA in 100ml of 10%TCA (10% TCA in distilled water) B.OTHERS Test tubes, Beaker, pipettes, burner, boiling waterbath etc. Procedure: 1. Pipette out differently 0.2-1ml of Std RNA solution and make up the volume upto 2ml by adding 10% TCA. 2. Use 2ml of TCA for the blank. 3. Add 3ml of Orcinol reagent in all the tubes, Mix well. 4. Boil the tubes in boiling water bath for 10 mins and cool it. 5. Take the OD at 665nm. Observation: Students are expected to write the protocol and observation of this experiment.

Result: The concentration of the given unknown was found to be-------- mg/ml .

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Experiment No: 11
Aim : Fractionation of Protein salt precipitation, solvent precipitation, iso-electric precipitation Salt Precipitation Principle: the charges on a protein in solution can also be neutralized by the addition of salts and this also has been used for purification of proteins. Theoretically any salt can be used but generally ammonium sulphate is preferred for two reasons . one, it has a high solubility , 840 g/liter, and secondly , its dissolution in water is exothermic or the solution gets cooled. Method : carry out the experiment given in the flow chart Estimate the amount of protein in each fraction and calculate back to per gram tissue weight. Care must be exercised to carry out the protein estimation, as ammonium sulphate will interfere with the assay. Therefore, it is preferable to adopt the modified biuret method after TCA precipitation. Alternatively, each fraction can be dialyzed to remove the salt and then estimation carried out. Solvent precipitation Another method of neutralizing the protein in solution and precipitating them out is by adding solvents like acetone or alcohol. Method : fractionate a tissue homogenate into different proteins by adding different amounts of ice cold acetone ( 30%, 40% and 50%). Tabulate the results. Isoelectric precipitation Take about 10 ml of milk in a beaker. Slowly add 1N acetic acid in drops. At a particular stage a sudden flocculent precipitation takes place. Measure the PH. It will be found to be about 4.5. the major protein of milk is casein and its isoionic point is 4.5. At this PH, the net charge on the molecule is zero. Remember that proteins remain in the solution mainly because of the charges present on them which makes them hydrophilic, once this charge is neutralized, the protein precipitate out. This is how cured is prepared. The inoculum added contains lactobacilli which utilize the lactose of milk to produce lactic acid. When the PH reaches 4.5, casein is precipitated out.

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Experiment No: 12
Aim : Demonstration of starch hydrolysis by given bacterial culture Principle : starch ( C6 H6 O5)n is an insoluble polymer of glucose which acts as a source of carbon for microorganisms which have an ability to degrade them. Starch degrading microorganism transport the degraded form across the cytoplasmic membrane of the cell. Some bacteria posses the ability to produce amylase that breaks starch into maltose. The amylase is an extracellular enzyme which is released from the cell of the microorganisms. Requirements 1) Bacterial culture 2) Inoculation loop 3) Starch agar medium* 4) Petri dish 5) Iodine *Starch Agar medium starch 20 g Beef extract 3 g Peptone 5g Agar 15 g Distilled water 1 liter Iodine solution ( as used in gram staing) Method a) Prepare starch agar plate and streak with suitable culture. b) Allow the microbe to grow at 37oC for 48 hours. c) Pour iodine solution in the plate. d) The blue-black colour appear due to formation of starch-iodine comples. If the area around streaked culture remain clear it indicates the degradation of starch has occurred due to production of amylase. Results The starch and iodine make a complex of blue colour. Iodine does not react with maltose or with any other product of starch degradation. Hence, no color is formed in such cases and clear area is visible. The given bacterial culture has been found to amylase positive Or The given bacterial culture has been found to be amylase negative.

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Aim-

Demonstration of amylase production by Aspergillus niger

PrincipleStarch is broken into monomer units by the enzyme amylase produced by the organism. However starch reacts with iodine and develops blue coloured complex. Moreover, intensity of the colour developed is directly proportional to the concentration of starch present in the sample. RequirementsGrowth Medium Yeast extract Malt extract Peptone - 0.5g Soluble starch Distilled water pH = 6.0 Reagents Iodine solution - 0.01N Starch solution - 0.1% Procedure1. Prepare growth medium, dispense 200 ml medium in flask and autoclave it. 2. Prepare pure culture of Aspergillus niger and prepare fresh culture in tubes. 3. Transfer 10ml of inoculum in sterilized growth medium and incubate at 30C for 24 to 48 hours. 4. Filter the filterate through sterile Whatman filter paper no.42. Collect supernatant and measure amylase activity of filterate by starch iodine method. 5. Take different aliquots of starch solution ranging from 0-2ml and make initial volume to 16ml by adding distilled water. Blank will lack starch. 6. Add 4ml of 0.01N iodine solution. Measure O.D after 10min of incubation at 578nm. 7. Draw graph between O.D and starch concentration. ObservationStudents are expected to write the observation of this experiment, which will be based upon the findings of the experiment. Result- 0.3g - 0.3g - 1.0g - 1000ml

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Aim : Demonstration of Protein (Gelatin) hydrolysis Principle: Gelatin is a polymer of amino acids and the protein is used as nitrogen and carbon source for microorganisms. The gelatin or protein is generally broken down into peptides of short amino acid polymers and amino acids can be transported into the cell. The enzyme that acts on or degrade the protein is called protease. The property gelatin is to remain solid below 22oC, while the degraded form of gelatin i.e. amino acids and peptides is to remain liquid below 22oC Requirements a) bacterial cultures b) trichloroacetic acid c) Nutrient gelatin broth/ agar d) Ice flakes e) Incubaors Nutrient gelatin broth/ agar medium: Gelatin 120 g Beef extract 3g Peptone 5g Distilled water 1 liter Agar ( if required) 15 g Method 1) prepare gelatin agar medium and streak the plates with given bacterial cultures. 2) Incubate the streaked plates at 20 oC for about a month and check for gelatin liquefaction. If gelatin is used as liquid. Inoculate and incubate the tubes at 35oC 3) After a month keep in ice to check liquefaction. 4) The third possibility is to use solidify medium and incubate the plates at 35oC 5) Flooded the plates with trichloroacetic acid Result Trichloracetic acid will precipitate the gelatin and plate will become opaque. In the same way casein hydrolysis can be performed. Growth of protease producing bacteria results in hydrolysis of casein by forming clear zone around the colonies.

22

Aim : Demonstration of Fat hydrolysis (lipase activity ) by a bacterial culture Oil is used as substrate. Some microorganisms have the ability to hydrolyse fats and results into rancidity in some food products. The ability of organisms to hydrolyse fat is accomplished due to the enzyme lipase. Fat molecules are degrade resulting in glycerol and fatty acids. Lipases are one of the most important classes of industrial enzymes. Many important biotechnological applications have been explored in paper industry units, organic chemical processing and agrochemical industry. Lipases have been used in food, dairy , beverages, and detergent formulation. Requriments a) Bacillus species b) Lipase producing broth* c) Nutrient agar medium d) Tween 80 (1%) * lipase producing broth medium for Bacillus Species NaCl CaCl2 Yeast extract Tween 80 (1%) Distilled water PH Method 1) 2) 3) 4) 5) grow 6 hour old culture if Bacillus sp. On nutrient agar slants containing 1% Tween 80. Add inoculum in flask containing lipase producing broth medium. Incubate the flask for 15 hours at 50 oC under continuous shaking condition i.e. 200 rpm. Centrifuge the contents at 1000 g for 20 minutes at 4 oC Meaure enzyme activity in cell free supernatent using p-nitrophenol palmitate as substrate by colorimetric assay. 5g 0.05 g 5g 5ml 1 liter 8.0

Result Express the result in terms of units ( U). one unit of activity is defined as the amount of enzyme releasing 1mg of p-nitrophenol/minute.

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Aim : Demonstration of Uease Production i.e. urea Hydrolysis Principle: urea is the waste nitrogenous material and excreted out by mammals. Some bacteria degrade the urea into ammonia and CO2. due to production of ammonia , the urease production can be easily demonstrated by the following reaction: NH2 CO2 - NH2 Requirements a) b) c) d) Proteus vulgaris, E. coli and Serratia marcecsens Urea broth medium* Inoculation tube Incubator 2 NH3 + CO2

* Urea broth medium urea yeast extract K2HPO4 K2HPO4 Phenol red Distilled water PH 20 g 0.1 g 9g 9.5 g 0.01 g 1 liter 6.8

Method 1) Incubate the urea broth medium with bacterial culture 2) Incubate the culture at 37oC for 48 hours. 3) The phenol red indicator will turn to pink due to alkaline nature of the medium of ammonia production as seen in case of P. vulgaris. Otherwise, indicator will remain yellow at acidic range of PH. then it shows no urease production by the given microorganisms as in case of E. coli and serratia marcescens. Results P. vulgaris can be distinguished by E. Coli and S. marcescens by its ability to produce large amount of the enzyme urease.

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Experiment No: 12
Aim: - To estimate the activity of - amylase. Principle: - -amylase E.C. No. 3.2.1.1 i.e endo 1,4 glucan hydrolase. It occurs widely in bacteria and fungi. All amylase are endo active enzymes which specially cleaves 1,4 glycosidic linkages in amylose , amylopectin and glycogen yielding sugar in configuration. They are unable to hydrolyse 1-6 branch points in amylopectin but are able to bypass this branch point. Two types of microbial amylase are existing i.e. saccharifying amylase and liquefying amylase. They are distinguished by the fact that saccharifying amylase produce a increased quantity of reducing sugar about twice of liquefying amylase.

Properties Molecular wt. Optimum pH Optimum temperature Metal ion req. Km(milimoles) Specificity Product Substrate Nature

lpha amylase 50,000 3.5 -7 35 -90 C Ca++ 1 Alpha 1,4 linkages can bypass alpha 1,6 linkages Maltodextrin Amylase,glycogen amylopectin Endosplitting

Beta amylase 55,000 1,60,000 5.5 -7.5 37 -55C 0.2 Alpha 1,4 limkages cannot bypass alpha 1,6 linkages Maltose and beta unit dextrin Amylase, glycogen, amylopectin Exosplitting

Amyloglucosidase 50,000 1,12,000 4.0 -6.0 40 70C -18.5 Alpha 1,4 and alpha 1,6 linkages Beta glucose, amylase and amylopectin glycogen, amylopectin,dextrin maltose. Exosplitting

25

Assay: - amylase are generally quantified by beta amylation of reducing group formed due to hydrolysis of soluble starch. The simplest method is dinitrosalicyclic acid method. Starch iodine colorimetric method is also used.. amylase: - amylase i.e. 1-4 1-4 glucanmalto hydrolase E.C. No. 3.2.1.2. It is a saccharifying amylase i.e. widely distributed in plants and also in microorganisms. amylase are exosplitting enzymes which attacks amylase , amylopectin, glycogen at non reducing terminal resulting in formation of maltose in configuration, amylases is specific for , 1-4 linkages and is unable to bypass 1,6 branch point. Assay: - Since the main product for amylase hydrolysis are the disaccharides maltose units , unit dextran, the reducing sugar assay described for alpha amylase may be employed. Amyloglucosidase E.C. No. 3.2.1.3 also called glucoamylase. Amyloglucosidase are exosplitting amylopectinamylopecic enzyme which attack amylase, amylopectin and glycogen are specifically hydrolysed 1, 4 and 1, 6 linkages from non reducing end. However 1,6 glucosidic linkages are cleaved by less readily than 1,4 glucosidic linkages. The product of reacton is glucose. Assay: - The major product of amylase action is glucose. The spectrometric assay for glucose may be employed. Procedure: - Take 0.5 ml of amylase enzyme in a test tube and 1 ml of buffer and 0.5 ml of starch. The glucose production in this tube is to be determined. Incubate these tubes for 10 min at 37 C then add 1 ml of DNSA reagent in it. Boil test tube for 10-20 min in water bath. A control tube is also performed in which DNSA is added before addition of the starch so, as to deactivate the enzyme. Arrange the test tubes as per protocol. After addition boil these test tubes for 10 15 min. Optical density of these tubes are to be read at 520 - 540 nm in colorimeter. Protocol: Reagents Starch Buffer Enzyme NaOH Enzyme DNSA C1 (ml) E1 (ml) C2 (ml) E2 (ml) C3 (ml) 2.5 2.5 3.5 3.5 4.5 3.5 3.5 2.5 2.5 1.5 1 1 INCUBATE FOR 30 MIN ONLY FOR EVP. 1 1 1 1 1 1 1 1 1 1 1 1 1 E3 (ml) 4.5 1.5 1 1 1 C4 (ml) 5.5 0.5 1 1 1 E4 (ml) 5.5 0.5 1.0 1 1

OBSERVATION: 1 OD(450nm) 2 3 4 5 6 7 8 9 10

Result: - The activity of amylase was found to be

g/ml from the graph.

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Experiment No: 13
Aim: - To study the effect of pH on activity of amylase. Theory: - Enzyme is a protein, every protein exists in active state at an optimum pH If pH is less than or greater than this optimum pH the enzyme will Undergo structural change which will decrease its activity. Thus if graph is plotted, a bell shaped curve is obtained; showing pH optimum at the top with gradual decrease of enzyme activity on either side. Procedure :- 1) Take 7 clean and dry test tube and label them for control, 1-6. To these add 1ml of buffered starch. 2) Now from 1-6 add different pH solution starting from 5.5-8 Respectively. 5ml of pH solution is added. 3) Now add 0.5ml enzyme solution in test tube from 1-6. 4) In the control add 5ml of any buffer and then add 0.5 ml enzyme Add 0.5ml NaOH so that reaction is terminated. 5) Now keep for incubation at 300C for 20min. 6) Now add 0.5ml NaOH in the tubes labeled 1-6, then add 1ml of DNSA solution in all the tubes and keep for boiling in water Bath for 15min. 7) Take the O.D after cooling the tubes at 546nm. Reagents: 1) Phosphate buffer (0.1M) - Add 40ml 0.2N NaOH to 50ml of 0.2M NaH2PO4 and dilute to 100ml. 2) Starch solution 1% in phosphate buffer 3) Enzyme solution -1% in bufferd saline 4) DNSAreagent. 5) pH variation- prepare pH from 5.5-8 by the help of a ph meter. The solutions used are 0.2M NaH2PO4 and 0.2M Na2HPO4 is mole toward acidic and it has a pH Around 4.5 .so with proper addition of discreat pH of 5.5, 6, 6.5,7,7.5,8 is obtained.

Result: - As the pH increase, activity of enzyme also increase, it reaches the maximum pH 6 after that the activity gradually decrease.

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Experiment No: 14
Aim: To study the effect of temperature on activity of enzyme ( amylase). Principle: -Amylase catalyses the hydrolysis of -1, 4 linkage of starch with production of reducing sugars .The reaction is followed by measuring the increase or decrease in the reducing sugars using DNSA reagent. As the temperature increases the activity of the enzyme initially increases, attains a maximum and then gradually decreases thus produces a bell shaped curve. REAGENTS: 1. Buffered starch-1% in phosphate buffer 2. Enzyme solution -0.5% in buffered saline 3. DNSA reagent -5g DNSA reagent in 100ml of 2M NaOH. Add 250ml of 150g Na-K tartarate . Make up the volume to 500ml with distilled water. 4. Phosphate buffer (0.1M): add 40ml 0.2 N NaOH to 50ml of 0.2 M NaH2 PO4. Mix well make up the volume to 500ml with distilled water. 5. 0.9% NaCl Procedure: 1. Take 7 clean and dry test tubes and label them as control and rest as 1 to 6. 2. To these tubes add 2.5ml starch solution followed by 1.5ml buffered saline. 3. Add 0.5 ml of 0.5 % enzyme solution to all the experimental tubes. 4. Prepare 6 water baths equilibrated at 5C, 15 C, 25 C, 35 C, 45 C, 55 C. 5. Incubate 1-6 tubes in respective water bath 6. Control can be incubated at room temperature or a separate control for each water bath is prepared. 7. Incubate for 20mins and then add 0.5ml NaOH in each of the experimental tubes 8. In control enzyme is added only after the addition of NaOH. 9. Add DNSA to each tube including control 10. Boil for 15 mins and read at 540nm. Observation: TEMPERATURE OPTICAL DENSITY(540nm) 30 C 45 C 60 C 75 C 90 C 105 C

Result: As the temperature of the solution increases the activity of the enzyme also increases at 75 C it shows optimum activity then as the temperature further increases the activity declines.

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Experiment No: 15
Aim:- Effect of inhibitor on enzyme activity. Principle- Inhibitors are classified into reversible and irreversible .Reversible inhibition are competitive or uncompetitive that can be effectively studied using bitterguard (karela). TUBES C1 E1 C2 E2 C3 E3 C4 E4 O.D 1.COMPETITIVE INHIBITION It is characterized by decrease in enzyme activity and increase in Km value, Vmax remains unaltered. Vo =Vmax[S]/Km(1+I/Ki) +[S] 2.NONCOMPETITIVE INHIBITION It is characterized by decrease in Vmax , Km being the same. Vo=Vmax/(1+I/Ki)[S]/Km+[S] 3.UNCOMPETITIVE INHIBITION- It is characterized by modification of both Km and Vmax. Vo= Vmax/(1+Io/Ki)[S]/[So]+Km(1+Io/Ki) REAGENTS- 1. Source of inhibitor:- a. Bitterguard pulp b. Seed pulp 2.Phospate buffer:- Add 40ml 0.2N NaOH to 50ml 0.2 NaH2PO4 and dilute it to 100ml. 3. Starch solution . 4. Enzyme solution 5. DNSA Procedure:- As per the protocol
REAGENT EN YME BUFFER INHIBITOR NaOH C1(ml) 0.5 1.0 1 C2(ml) 0.5 0.8 0.2 1 C3(ml) 0.5 0.6 0.4 1 C4(ml) 0.5 0.4 0.6 1 E1(ml) 0.5 1.0 E2(ml) 0.5 0.8 0.2 E3(ml) 0.5 0.6 0.4 E4(ml) 0.5 0.4 0.4 -

Boil for 10 min. in waterbath and read at 540nm

STARCH NaOH DNSA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Incubate for 15min. at 37C .

Boil for 15min.in waterbath at 540nm. Observation:Result:-

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Experiment No: 16
Aim: To determine the effect of substrate concentration on enzyme activity. Principle:- Amylase acts on starch to produce glucose. In this particular experiment, the amount of starch i.e the substrate is kept constant where as the concentration .of amylase is taken in increasing order. It is observed that the amount of glucose produced in every successive tubes also increases. This is due to reason that as the enzyme concentration increases the number of active site also increases. Thus substrate readily binds to enzyme thereby increasing the amount of glucose produced. Thus increase in concentration of enzyme increases activity of amylase. Care should be taken that:1) Amount of substrate should be in excess, so that becomes rate-determining factor. 2) All other parameters like time, PH and temperature should be kept constant. 3) Tubes should be clean and dry to avoid contamination due to inhibition and dilution by reagents. Procedure:1) 2) 3) 4) 5) 6) 7) 8) Take 7 clean and dry test tubes and label them, add 2.5 ml of starch solution. Add gradually increasing concentration of enzyme solution in tube 1-6 as per protocol. Incubate tubes for 30 mins. After incubation add 0.2N NaoH to each tube. Add 1ml of DNSA to each tubes. Keep in boiling water bath for 15mins and cool it. If required dilute it to 10ml with distilled water. Read OD at 590nm.

Observation Table:C1/E1 Optical Density C2/E2 C3/E3 C4/E4

Result:-

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Experiment No: 17
Aim: Evaluation of KINETIC CONSTANT [Km and Vmax] of purified enzymes. Principle: When all the other parameters including enzyme concentration, time and temperature are kept constant and the initial substrate concentration is varied between wide limits, the changes in the rate of enzyme-catalyzed reaction may be described in the following fig:

When more than one substrate are involved then, the concentration of second substrate should also be kept constant. As the concentration of substrate increases the rate of reaction is also increased upto a certain limit. This is apparent from the initial linear curve with further increase in substrate concentration. Thus linearity does not persist as the increase in rate of reaction gradually goes on decreasing, finally when the rate of reaction is not affected by increase in substrate concentration the curve shows plateau. The velocity indicating this splitting is called as Vmax. Km is substrate concentration at half the Vmax. REAGENTS: 1. Phosphate buffer 2. Enzyme 3. Starch solution 4. DNSA 5. Sodium hydroxide (NaOH)

31

Procedure: Arrange the test tubes and perform according to the protocol. Sr no. 1 2 3 Reagents Enzyme Phosphate buffer Starch (1%) NaOH Enzyme DNSA control 1 4 1 0.5 4.8 0.2 2 0.5 4.6 0.4 3 0.5 4.4 0.6 4 0.5 4.2 0.8 5 0.5 4.0 1.0 6 0.5 3.8 1.2 7 0.5 3.5 1.5 8 0.5 3.0 2.0 9 0.5 2.5 2.5 10 0.5 2.0 3.0

INCUBATE FOR 20 MINS 4 5 6 1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Boil in water bath for 15 mins and cool .Take the OD at 450nm. Observation: 1 OD(450nm) 2 3 4 5 6 7 8 9 10

Result: The value of Km was found to be

g/ml.

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Experiment No: 1
Aim:- Separation of green plant pigments by column chromatography. Principle:- Column chromatography using alumina gel works on the basis of physical factor of adsorption only and hence it is called as column chromatography. Adsorption is the phenomenon whereby a substrate gets bound to the surface of unique particle. If the compound is in the solution, particles insoluble in solvent then part of substrate is adsorbed and part remain in the solution. The ratio between amount adsorbed and amount remained in the solution is constant called adsorption coefficient. The extent of binding between solute and adsorbent depends on charge, van der Waals' forces, dipole interaction, hydrogen bonding and steric forces and depends upon the structure of compounds. The mass of solute adsorbed per unit weight of adsorbent [m] depends on the concentration of solute. According to Langmuir equationm = K 1K 2c 1+K2c Where, m = Mass of solute adsorbed per unit weight of adsorbent. K1= Number of active adsorption sites per unit and depend upon nature of Adsorbent. K2= Affinity of solute for adsorbent and depends upon component of system. Langmuir assumed only one binding site on the adsorbent. Hinshelwood equation for number of different binding sites is as followsm = K 1K 2c 1+K2C The molecule with least adsorbtivity goes along with the solvent and these are eluted first followed by other component. Materials :1) Chromatographic Column 2) Fresh Spinach Leaves 3) Alumina 4) Calcium Carbonate 5) Sodium Sulphate 6) Petroleum Ether 7) Methanol, Benzene, Waring Blender. Procedure:Preparation of extract: Homogenize 5 to 10gm of leaves in Waring blender adding 20 to 40gm mixture of petroleum ether, methanol and benzene in 45:15:5 proportions. Filter the extract through separating funnel. Add 10 to 20 ml of water, shake well and allow layer to separate. Remove the lower layer containing methanol. Repeat the addition of water, shaking and removing the aqueous layer 3 to 4 times till the lower layer becomes colorless and only faintly colored. Avoid the vigorous shaking to avoid emulsion formation. Remove the last traces of water by adding anhydrous sodium sulphate. Filter to remove the solid and concentrate the extract to few ml by careful evaporation up to dryness. If presence of H2O is suspected, repeat the above steps. II] Preparation of column:

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Alumina was used as adsorbent. Column was filled with 5 cm alumina, 7cm calcium carbonate, 7cm sucrose in petroleum ether. A small amount of glass wool is pushed down to the bottom as a pad. About 2 to 5 g of adsorbent [previously dried at 120oC overnight for activation] is taken in a beaker. A small amount of C6H6 added to slurry. The slurry is carefully poured into a burette; use more benzene if necessary to pour the extra slurry. The adsorbent should never be allowed to dry. After all the adsorbent has settled add 20ml of more benzene and allow it to pass through the column. When the solvent level is just about 1cm above the solid layer close the stop cork. III] Separation of solution of pigment: Take extract as prepared from (I) part in 1ml of benzene add quantitatively, transfer it with a pipette to the top of the column taking care not to disturb the surface of the layer. Open the stop cork and allow the solution to separate. When the level of the solvent has just reached the surface of adsorbent, a small amount of benzene is added to develop the column. It is likely that one or two yellow bands will appear and move down the column, but most of the material will not remove from the top. This is because benzene is not polar enough to release all adsorbed compound when 20ml of benzene has just been run then, add 5ml of 5%acetone in benzene from the top. Note the changes by continuously increasingly the acetone concentration till only pure acetone is added. Collect the fraction by taking it in test tube below the burette and plot the adsorption spectrum of each colored peak. Note : The observation and result should be written as given below. OBSERVATION:Three colored layers were observed. Sr. no. 1. 2. 3. Pigments Chlorophyll Xanthophylls Carotenoids Color of bands Green Yellow Orange

RESULT: - The three colored layers of chlorophyll (green), xanthophylls (yellow) and Carotenoids (orange) were separated by column chromatography.

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Experiment No: 1 -21

Aim: To calculate the mean ,median ,mode of the given problems. I.Calculate the arithmetic mean for following data. Class Interval 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 frequency 2 7 17 29 29 10 3 2 1

Arithmatic mean :It is sum of all data divided by number of observation . Arithmatic mean for Ungrouped data = X = X1+X2+X3+Xn n x =sum of all the value of observation n = total number of observation . Arithmatic mean For grouped data = X = fx
n

f =frequency i.e number of repeatition II. Calculation mode of following data and median. Age of students: 10, 12 15 ,20,20,22,22,15,25,15,14,19,20,15,15 MODE: It is the maximum frequency observation i.e the value which is repeated Maximum times in the given data. MEDIAN : When the data is arranged in ascending or descending order the Median value is called median. 1. If observation are odd Median = [ n+1 / 2 ]th 2.If the observation are even Median = [ n /2 ]th +[n+2/2]th/2 Result: From Calculation: Mean is found to be - 48.40 Mode is found to be -15 Median is found to be -15 Note: The values given here are for better understanding of student but lecturer should give to the student different values for practice.

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Experiment No: 22
Aim: To find out whether the dihybrid ratio is good to fit or not in the following two crosses from the data given using 2 test.

Principle: When stastical test is used to compare on observed ratio or value with an Expected or theoretical ratio or value and determine how clearly the former fits into latter. It is known as Testing the goodness of fit One measure commonly used for it 2 test.[Chi-squareTest] { [Observed] - [Expected] }2

Expected value To accept or reject Null Hypothesis, this calculated 2 is compared with standard Value . If the value of 2 is less than that of table 0.05 probability , than Null Hypothesis is accepted. Di-hybrid Cross: When the cross is made between two pair of contrasting character, it is said to be dihybrid ratio RRYY and rryy. When plants with the above character were crossed pollinated in F1 generation they produced hybrid plan [Rr Yy] with round and yellow seed. When these (F1) plants were allowed to self pollinate, progeny were formed in (F2) generation in the ratio 9:3:3:1

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Round Tellow Round Green

9 3 3 1

Wrinkle Yellow Wrinkle Green -

The Mendelian cross ratio is expected for every dihybrid cross [Null Hypothesis] The ratio (F2) as cited above is actually based on Law of Independent Assotrment Sometimes if more than 2 genes are located on same chromosome independent assortment does not occur, instead they may be inherited together in a group. this process is called linkage. The Mendelian ratio varies in such cases Alternate Hypothesis. Result: From the above observation, data obtained from cross I is supported statistically, To be dihybrid cross [Accept Null Hypothesis] While data obtained from cross II is supported statistically not be dihybrid Cross [Accept Alternate Hypothesis] Note: The values given here are for better understanding of student but lecturer should give to the student different values for practice.

37

Experiment No: 23
Aim: A bag contains 10 pink,10 yellow,10 orange and 20 red beads . III. IV. What is probability of drawing 1 yellow bead . What is probability of drawing 1 yellow and 1 red bead.

Theory: If an experiment has an m mutually exclusive equally , likely and exhaustive cases , out of which m are favourable to the happening of the event A , then the probability of the happening of A is denoted by P(A) and is defined as:-

P(A) = m n = no. of cases favorable to A Total[exhaustive] no. of cases Probability of an event which is certain to occur is one and the probability of an impossible event is zero . The probability of occurrence of any event lies between zero & one [0-1] , both inclusive. Procedure: 1. Withdraw a single bead at randomly from a mixture of 50 beads [yellow +pink+red] 2. 3. 4. 5. Record the observation of withdrawal of yellow bead. Withdraw one yellow and one red bead at a time from a mixture of 50 beads and the observation of it. In the first case beads one withdrawn 50 times and in second case beads are withdrawn 25 times. Calculate the probability.

Observation Table:Sr no. 1. 2. Beads Yellow Yellow+ red Tally Frequency 15 5 Probablity 3/10 1/5

Calculations:1. The total number of equally , likely and exhaustive case = n = 10+10+10+20= 50 No. of favourable cases = m = 10 probability of drawing = m = 10 = 1 n 50 5

38

1 yellow bead Practically numbers of favourable case (m) =15 probability of drawing of = m =15 = 3 n 30 10 1 yellow bead 2. The total no. of equally , likely an exhaustive case = n = 10+10+10+ 20 = 50 Number of favourable case (m1) Probability of drawing one yellow and one red bead together = 10 * 20 50 50 Practically number of favourable cases m = 5 Result : i) ii) The probability of drawing a yellow bead is theoretically 1/5 and practically it is 15/50 Probablity of drawing of yellow & red bead together is theoretically 2/25 practically 5/25. and = 2 25

Note: The values given here are for better understanding of student but lecturer should give to the student different values for practice.

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Experiment No: 24
Aim: Calculate standard error (sampling error) from the observation obtained by drawing samples randomly for 25 times of sizes 5 beads at a time from population for getting pink beads. Theory: Standard error = Standard deviation No of observations Requirements: Beads of different colors essentially having pink colored beads Procedure: 1. From the population of 50 beads, withdraw 5 beads randomly at a time. 2. Calculate the number of pink beads in each withdraw. 3. Withdraw the beads 25 times. 4. Calculate the mean for the observation of pink beads. 5. Calculate its mean deviation and then calculate standard error. Observation Table:
Sr.no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Sample drawn 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Number of pink beads 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 x- x 32 30 31 32 32 31 32 29 29 30 32 30 30 31 31 30 31 32 30 32 29 30 31 28 32

Where, x=32 x- x = 768 Result: Standard error was found to be = 4718.5. Note: The values given here are for better understanding of student but lecturer should give to the student different values for practice.

40

Equipments generally required in the Biotechnology laboratory

Micropipettes

Micropipettes kept in micropipette stand

Petriplates with different media

Binocular Microscope

41

Colorimeter

Laminar Air Flow

Spectrophotometer

Waterbath

Incubator

Spectrophotometer

42

Distilled Water Assembly

Mono-pan balance

Single-pan manual balance

Centrifuge

43

Autoclave

Distilled Water Assembly

Tipbox

Incubator Shaker

44