Cell and Molecular Biology Lab-06BTL47

INSTRUCTIONS
Reading the warning signs (hazard symbols) and labels, while working in the laboratory. Lab coats or ankle length aprons must be worn while handling toxic, corrosive and flammable materials. Gloves should be worn, while handling corrosive and highly toxic chemicals. Appropriate eye protection should be worn at all times in laboratories. Always wash hands with soap after working with chemicals, even though gloves have been used. Do not mouth pipette or siphon toxic chemical reagents, corrosive liquids, organic solvents, strong acids and alkalies. Use pro-pipette or an autodispenser for dispensing. Do not directly smell, sniff or taste any chemical. Avoid inhalation. Containers should be closed when not in use. When working with flammable chemicals, make sure that there are no sources of ignition near by, in order to avoid fire or explosion (like naked flame of Bunsen burner, electrical hot plate etc.). Handle toxic, corrosive chemicals and flammable solvents in a chemical safety hood or a fume hood. No smoking in any area of a laboratory. No eating, drinking of beverage or application of cosmetics in the laboratory, except in designated areas in which no chemicals are used or stored. Avoid working alone in the laboratory. If there are any questions about a procedure or the hazards of a chemical, ask the lab supervisor or the instructor before performing the procedure. Know the location and proper use of emergency equipment (like fire extinguisher, eye wash fountains) and First Aid Kit. Contaminated cultures should be autoclaved before disposal.

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Cell and Molecular Biology Lab-06BTL47

CONTENTS Sl.No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Name of the Experiment Study of divisional stages in Mitosis. Study of divisional stages in Meiosis. Study of Polytene and Lampbrush chromosomes. Isolation of plant protoplasts by enzymatic method. Chemical fusion of plant protoplasts (PEG, Calcium). Isolation of plasmid DNA from E.coli. Isolation of genomic DNA (plant/ animal/ microbial sources). Agarose gel electrophoresis and quantification of nucleic acids. (colorimetric, ethidium bromide dot blot and standard DNA marker) Plasmid gene mapping in E.coli Restriction mapping/ digestion of genomic DNA. Transformation of E.coli cells. Selection of recombinants in E.coli (Blue-white screening). Tns 5 induced mutagenesis in E.coli. Study of conjugation in E.coli. SDS-PAGE. PCR (Demo experiment). 27-28 29-30 31-32 33 34-35 36-37 38-43 44-45 Page No 3-5 6-8 9-10 11-13 14-15 16-20 21-24 25-26

STUDY OF DIVISIONAL STAGES IN MITOSIS

Objective: To study the different stages in mitosis Introduction:

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Cell and Molecular Biology Lab-06BTL47 Mitosis is a process of cell division which results in the production of two daughter cells from a single parent cell. The daughter cells are identical to one another and to the original parent cell.

Legend: Illustration of the process by which somatic cells multiply and divide.

Mitosis can be divided into four stages:

Prophase: The chromatin, diffuse in interphase, condenses into chromosomes. Each chromosome has duplicated and now consists of two sister chromatids. At the end of prophase, the nuclear envelope breaks down into vesicles.

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Cell and Molecular Biology Lab-06BTL47

Metaphase: The chromosomes align at the equitorial plate and are held in place by microtubules attached to the mitotic spindle and to part of the centromere. Anaphase: The centromeres divide. Sister chromatids separate and move toward the corresponding poles. Telophase: Daughter chromosomes arrive at the poles and the microtubules disappear. The condensed chromatin expands and the nuclear envelope reappears. The cytoplasm divides, the cell membrane pinches inward ultimately producing two daughter cells (phase: Cytokinesis).

Number of Chromosomes in some eukaryotic species

Organism Yeast (Saccharomyces cerevisiae) Slime mold (Dictyostelium) Arabidopsis thaliana Corn Onion Lily Nematode (Caenorhabditis elegans) Fruit fly (Drosophila) Toad (Xenopus laevis) Lungfish Chicken Mouse Cow Dog Human Chromosome numbera 16 7 5 10 8 12 6 4 18 17 39 20 30 39 23

Observation of permanent slides:

Observe the given permanent slides and identify the different stages of the mitosis. Draw a neat labeled diagram for each stage.

Staining procedures for chromosome analysis Objective: To observe different stages of mitosis in the onion root tip cells.
Materials required: Onion root tips Equipments: Waterbath, Microscopes, Spirit lamp Reagents: 45% Acetic acid. 4

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Cell and Molecular Biology Lab-06BTL47 Stain: 1% Aceto-Orcein: Dissolve 2 g of orcein in 100ml of 45% acetic acid by boiling gently. Mix the solution and filter. Dilute 1:1 with 45% acetic acid when ready to use. Others : Slides ,Cover slips ,Watch glass, Petri dishes. Collection and preparation of plant materials Root tips 1. Collect the root tips and immerse them in cold water. 2. Keep the root tips in a refrigerator at 20 C for 24 h. 3. Transfer the root tips to 3:1 fixative (3 parts ethanol: 1 part glacial acetic acid) for 1h before processing. 4. Place root tips in watch glass containing stain and keep the material for 1h 5. Place one root tip on a slide and add a drop of 45% acetic acid. 6. Remove the other areas of root tips except intensely stained region. 7. Cut the stained region into small pieces and place the cover slip over the solution. 8. Gently heat the slide over an alcohol flame and press out any excess acetic acid between layers of tissue paper. 9. Apply some pressure with the fingers to flatten the cells and observe the stages under microscope. Note: the best treatment to arrest cell division and accumulate metaphase cells varies for different species. Cold water treatment is best for Brassica and cereal species. For some other species, treat the roots in a 2 mM aqueous solution of 8-hydroxyquinoline at room temp for 4h, then add an equal volume of cold 0.1 M colchicines, and place in the refrigerator overnight at 20C. Observation: Observe the different stages of mitosis in the onion cells.

STUDY OF DIVISIONAL STAGES IN MEIOSIS

Objective: To study the different stages in meiosis. Introduction:
Meiosis is the type of cell division by which germ cells (eggs and sperm) are produced. Meiosis involves a reduction in the amount of genetic material. Meiosis

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Cell and Molecular Biology Lab-06BTL47 comprises two successive nuclear divisions with only one round of DNA replication.

Legend: Illustration of the process by which a single parent diploid cell (Both homologous chromosomes) divides to produce four daughter haploids cells (One homologous chromosome of the pair).

Four stages can be described for each nuclear division.

Interphase: Before meiosis begins, genetic material is duplicated. First division of meiosis
o

Prophase 1: Duplicated chromatin condenses. Each chromosome consists of two, closely associated sister chromatids. Crossing-over can occur during the latter part of this stage. The prophase 1 is divided into following stages

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Cell and Molecular Biology Lab-06BTL47 1. Leptotene: a stage of meiotic prophase immediately preceding synapsis in which the chromosomes appear as fine discrete threads. 2. Zygotene: the stage of meiotic prophase which immediately follows the leptotene and during which synapsis of homologous chromosomes occurs. 3. Pachytenee: the stage of meiotic prophase which immediately follows the zygotene and in which the paired chromosomes are thickened and visibly divided into chromatids. 4. Diplotene: a stage of meiotic prophase which follows the pachytene and during which the paired homologous chromosomes begin to separate and chiasmata become visible. 5. Diakinesis: the final stage of the meiotic prophase marked by contraction of the bivalents.
o o o

Metaphase 1: Homologous chromosomes align at the equatorial plate. Anaphase 1: Homologous pairs separate with sister chromatids remaining together. Telophase 1: Two daughter cells are formed with each daughter containing only one chromosome of the homologous pair. Prophase 2: DNA does not replicate. Metaphase 2: Chromosomes align at the equatorial plate. Anaphase 2: Centromeres divide and sister chromatids migrate separately to each pole. Telophase 2: Cell division is complete. Four haploid daughter cells are obtained.

Second division of meiosis: Gamete formation

o o o o

One parent cell produces four daughter cells. Daughter cells have half the number of chromosomes found in the original parent cell and with crossing over, are genetically different. Meiosis differs from mitosis primarily because there are two cell divisions in meiosis, resulting in cells with a haploid number of chromosomes.

Observation of permanent slides: Observe the given permanent slides and

identify the different stages of the meiosis. Draw a neat labeled diagram for each stage.

Staining procedures for chromosome analysis

Objective: To observe different stages of meiosis in the cells of immature anthers of onion.

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Cell and Molecular Biology Lab-06BTL47 Materials required: Onion root tips Equipments: Waterbath, Microscopes, Spirit lamp. Reagents: Ethanol, Chloroform, 45% Acetic acid (6:3:1) Stain: Aceto Carmine: Dissolve 1-2g of carmine in a solution containing 45 ml of glacial acetic acid and 55 ml of distilled water, boil gently, cool to room temp, shake, and filter the solution. Others : Slides ,Cover slips ,Watch glass, Petri dishes. Collection and preparation of flower buds. 1. Collect immature flower buds and store in fixative for 1 day at room temp. (Fixative: 6 parts ethanol: 3 parts chloroform: 1 part acetic acid). 2. Place the immature inflorescence directly into the stain. 3. Separate the anthers and placed on a slide in a drop of acetic acid or acetocarmine. Cut the anthers into small pieces and squeeze the anther segments. 4. Remove the debris, and place a cover slip over the solution. 5. Gently heat the slide to cause the cells to swell and the chromosomes to spread so that overlapping is minimized. 6. Apply some pressure with the fingers to flatten the cells and observe under microscope. Observation: Observe the different stages of meiosis in the onion cells.

STUDY OF POLYTENE AND LAMPBRUSH CHROMOSOMES.

Objective: To study Polytene and Lampbrush chromosomes. Introduction:

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Cell and Molecular Biology Lab-06BTL47 To increase cell volume, some specialized cells undergo repeated rounds of DNA replication without cell division (endomitosis), forming a giant polytene chromosome. Polytene chromosomes form when multiple rounds of replication produce chromatids that remain synapsed together in a haploid number of chromosomes. They have characteristic light and dark banding patterns which can be used to identify chromosomal rearrangements and deletions. Chromosome puffs (Balbiani ring) are diffuse uncoiled regions of the polytene chromosome that are sites of RNA transcription. In addition to increasing the volume of the cells nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as multiple copies of genes permits a high level of gene expression. Polytene chromosomes were originally observed in the larval salivary glands of Chironomus midges by Balbiani in 1881, but the hereditary nature of these structures was not confirmed until they were studied in Drosophila melanogaster in the early 1930s by Emil Heitz and Hans Bauer. They are known to occur in secretory tissues of other dipteran insects such as Malpighian tubules of Sciara and also in protists, plants, mammals, or in cells from other insects.

Fig. Polytene chromosomes in a Chironimus salivary gland cell

Observation: Observe the polytene chromosome.

Lampbrush chromosome
A Lampbrush chromosome (first seen by flemming) is a large chromosome (largest) found especially in the oocytes (immature eggs) of amphibians, birds and other animals. Lampbrush chromosomes occur during the diplotene stage of meiosis I. Lampbrush chromosomes are meiotic bivalents, each consisting of 2 sister chromatids. Each halve-bivalent is represented by two long strands that form many brushes like loops along the main axis of the chromosome. The outgrowths make DNA available for transcription during the maturation of the egg. Usually there is a little gene expression at meiosis, so it is not so easy to identify the activities of individual genes. Giant chromosomes in the lampbrush form can solve this problem,

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Cell and Molecular Biology Lab-06BTL47 since they allow the individual transcription units to be examined. Lampbrush and chromosomal puffs in a cell indicate that the transcription of tRNA is taking place.

ISOLATION OF PLANT PROTOPLASTS BY ENZYMATIC METHOD

Objective: To isolate intact protoplasts from given sample.

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Cell and Molecular Biology Lab-06BTL47 Introduction: Protoplasts are cells (plant, fungal or bacterial) that have had their cell walls removed. This can be done mechanically, or by digestion with enzymes. The naked cells are surrounded only by a cell membrane and can be used in a variety of ways. For example, two or more protoplasts can be fused with the help of a detergent, polyethylene glycol, to produce hybrid cells with characteristics from each parent. Infection of protoplasts with genetically-modified Agrobacterium tumefaciens is one way of introducing new genes into plant cells. Whole plants can be regenerated from protoplasts grown on solid or liquid media. The many advantages of the enzymatic method of isolation of protoplasts include: 1. Large scale reproducible isolation of protoplasts from various tissues. 2. Osmotic shrinkage is minimum and the deleterious effects of excessive plasmolysis are minimized. 3. Cells remain intact and are not injured as is the case of mechanical methods of isolation. 4. Protoplasts are readily obtained. The isolation and viability of protoplasts depend on a number of factors: age and physiological state of the plant, concentration and purity of the enzyme, pH, period of incubation in enzyme mixture, and the plasmolyticum.

Materials:

70% Ethanol 0.1% Mercuric Chloride 0.5M Mannitol Enzyme solution : 1% Cellulase 0.25% Macerozyme R-10 27.2 mg/l KH2PO4 101 mg/l KNO3 1480 mg/l CaCl2.2H2O 246 mg/l MgSO4.7H2O 0.5M Mannitol (pH 5.6) Cell Protoplast Washing Medium: 0.5M Mannitol (pH 5.6) 27.2 mg/l KH2PO4 101 mg/l KNO3 1480 mg/l CaCl2.2H2O 246 mg/l MgSO4.7H2O Modified Protoplast Culture medium (Table is listed below)

Procedure:

Take fresh leaves from 4-5 week old aseptically growing plants. Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute. Wash the leaves thoroughly with sterile distilled water. 11

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Cell and Molecular Biology Lab-06BTL47

Peel the lower epidermis of the leaf tissue (or make into small pieces) and place it in 0.5M Mannitol solution for one hour in a Petri plate. Remove the Mannitol solution and replace it with filter sterilized enzyme solution. Incubate the plates in dark for 16-20 hours at 25oC. Separate the protoplast layer by filtration using nylon membrane. Centrifuge the filtrate at 1000g for 10 minutes at 4oC. Remove the supernatant and resuspend the pellet in cell protoplast washing medium. Centrifuge at 1000g for 10 minutes. Repeat step 9 twice. Finally suspend the protoplast pellet at a density of 5x 105/ml in the modified protoplast culture medium. Plate the protoplasts as fine thin layer in Petri plates. Incubate the plates at 25o C in dark.

Modified Protoplast Culture Medium S.No.

Constituents
Mineral Salts Ammonium Nitrate Potassium Nitrate Boric acid Potassium Dihydrogen Phosphate Potassium Iodide Sodium Molybdate Cobalt Chloride Dihydrate Calcium Chloride Dihydrate Magnesium Sulphate Heptahydrate Manganese Sulphate tetrahydrate Zinc Sulphate Heptahydrate Copper Sulphate Pentahydrate Ethylene Diamino Tetra Acetic acid Disodium salt Ferrous Sulphate heptahydrate Sugars Glucose Ribose Mannose Sorbitol Organic Acids Sodium Pyruvate

Amount

A
1 2 3 4 5 6 7 8 9 10 11 12 13 14

82.5 g/l 95.0 g/l 1190 mg/l 34 mg/l 166 mg/l 250 mg/l 25.0 mg/l 88.0 gm/l 74.0 gm/l 4460 mg/l 1720 mg/l 25.0 mg/l 7.45 g/l 5.57 g/l

B
1 2 3 4

30.0 g/l 0.5 g/l 0.5 g/l 0.5 g/l

C
1

5.0 mg/l

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Cell and Molecular Biology Lab-06BTL47 2 3 Citric acid Malic acid Vitamins Thiamine Hydrochloride Nicotinamide Pyridoxine Hydrochloride Calcium pantothenate Biotin Ascorbic acid Riboflavin Inositol Hormones 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) Benzyl Amino Purine (BAP) 0.5 mg/l 1.0 mg/l 100 mg/l 500 mg/l 500 mg/l 250 mg/l 100 mg/l 75.0 mg/l 50.0 mg/l 100 gm/l 10.0 mg/l 10.0 mg/l

D
1 2 3 4 5 6 7 8

E
1 2

Observation: Observe the isolated protoplasts

Pre-digestion

Post-digestion

Protoplasts

CHEMICAL FUSION OF PLANT PROTOPLASTS (PEG, CALCIUM)

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Cell and Molecular Biology Lab-06BTL47

Objective: To produce somatic hybrids by the fusion of protoplasts Introduction: Somatic hybridization is one of the most important use of protoplast culture. This is particularly significant for hybridization between species or genera. This cannot be made to cross by conventional method of sexual hybridization. During the last two decades, a variety of treatments have been successfully utilized for fusion of plant protoplasts. These treatments particularly include the high pH with high Ca++ ion concentration, polyethylene glycol and NaNO3 treatment. Materials: Intact protoplasts of two different species, culture medium, CaCl2 2H20 Solution, PEG, Petri plates. Procedure: A. Treatment with Calcium ions (Ca++) at high pH:1. Spinning (Centrifugation) the protoplasts in a fusion inducing solution (0.05M CaCl2 2H20 in 0.4M mannitol at pH 10.5) for 30 min. at 50g. 2. After centrifugation, the tubes are placed in a water bath (37 0c) for 4050 min. This leads to fusion of 20-50% of the protoplasts. Procedure: B. Treatment with polyethylene glycol. The following procedure is followed when protoplasts are available in sufficient Quantity 1. 1ml of culture medium with suspended protoplasts is added to 1ml of 56% of solution of PEG, and tubes shaken for 5 Sec. 2. The protoplasts were allowed to settle for 10min, washed with growth medium and examined for successful agglutination and fusion. The following procedure is followed when protoplasts are available in 1. 2. 3. 4. micro quantities . Two types of protoplasts are mixed in equal quantities an 4-6 micro drops (100microliters each) are placed in small Petri plate and allowed to settle for 5 to 10 min at room temperature. 2-3 drops of PEG are added from periphery in each Petri plate which is Incubated 30min at room temperature. After PEG treatment, protoplasts are gradually washed in medium ( during this process most of the fusion is achieved). PEG is then replaced by culture medium to allow the growth of fused protoplasts.

This leads to agglutination of protoplasts. Some times a cover glass is placed in the middle of Petri plate before protoplast suspension is poured. This avoids sticking of protoplasts to the floor of Petri plate and also makes its convenient to handle the protoplasts including their fixation, staining and examination. After PEG treatment, protoplasts are gradually washed and during this process most of the fusion is achieved. Observation: Fused protoplasts were observed in the medium. 14

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Cell and Molecular Biology Lab-06BTL47

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Cell and Molecular Biology Lab-06BTL47

ISOLATION OF PLASMID DNA FROM ESCHERICHIA COLI

Objective: To isolate the plasmid DNA from E.coli cells by alkaline lysis method. Introduction: Plasmids are double stranded circular DNA molecules that have a property of self replication, independent of bacterial chromosomal DNA. The American Molecular biologist Joshua Lederberg was the first to introduce the term plasmid. The size of the plasmids varies from 1-400 kilobasepairs (Kbp). There are five main classes of plasmids: Fertility-F- plasmids, Resistance-R- Plasmids, Col plasmids, degradative & virulence plasmids. They serve as important tools in genetics and biochemistry labs, where they are commonly used to multiply or express particular genes. Though the presence of a plasmid in a bacterial cell is detected genetically as a change in phenotype of the bacteria, it is often necessary to isolate plasmid DNA for molecular studies such as size determination, restriction enzyme mapping, nucleotide sequencing or for construction of new hybrid [plasmids, PCR, expression of proteins, transfection and gene therapy. The plasmid DNA of the bacteria are closed circular molecules of double stranded DNA that range in size from 1Kb to more than 200Kb. They are found in bacteria as additional hereditary units and replicated independently of bacterial chromosome. However, the plasmids rely upon enzymes and proteins from their host for their successful transcription and replication. Plasmid are useful to bacterial cells because they can carry genes that code for enzymes which may be involved in resistance to antibiotics, toxins in non ideal environmental conditions, or production of toxins within the bacteria itself. Principle: Isolation of plasmid DNA from E.coli is the most common and routine procedure in research laboratories. There are many methods to isolate plasmids from bacteria. The widely practiced procedure is the alkaline lysis of cells. This protocol is often referred to as mini-prep, and yields fairly clean DNA, and easily. This step involves three steps i) Growth ii) Harvest and Lysis iii) isolation of plasmid. Bacterial cells are first grown with antibiotic for maintaining the plasmid within the cells. Without this selective pressure, the bacteria tend to lose the plasmid and will not replicate the extra chromosomal plasmid if it is not needed. The resuspension buffer (minimizes the physical forces involved in lysis) contains glucose, TRIS, EDTA and RNase. Glucose is added to increase the osmotic pressure outside the cells. Tris is used to maintain a constant pH (= 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); by binding to divalent cations that are necessary for DNAse activity. RNase degrades the RNA. The solution II contains NaOH and SDS (a detergent). The alkaline mixtures rupture the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands. Solution III contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. Strands of covalently closed circular plasmid DNA renatures immediately when the conditions return to normal. The E.coli chromosomal DNA, also partially gets renatured tangle at this step, which is trapped in the precipitate formed by the potassium acetate and SDS from solution, along with the cellular debris. Most of the chromosomal DNA and bacterial proteins precipitate along with SDS complex and is removed by centrifugation. The plasmid DNA remains in solution. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick Department of Biotechnology, CMRIT, Bangalore-37. 16

Cell and Molecular Biology Lab-06BTL47 precipitation can therefore purify DNA from protein contaminants. Ethanol helps to remove the remaining salts and SDS from the preparation. Materials Required: Medium: LB broth Culture: 24hrs of culture E.coli Reagent: Solution I: GTE buffer: 50 mM glucose -9 ml 50% glucose 25 mM Tris-HCl pH 8.0 -12.5 ml 1 M Tris-HCl pH 8.0 -10 ml 0.5 M EDTA pH 8.0 10 mM EDTA pH 8.0 RNase -100g/ml Make up the volume to 500 ml with distilled water Solution II: 0.2N NaOH/ 1% SDS 1% SDS -50 ml 10% SDS 0.2 N NaOH -100 ml 1 N NaOH Make up the volume to 500 ml with distilled water Solution III: 5M Potassium acetate: 29.44g potassium acetate, 11.5ml acetic acid, make up to 100ml with distilled water. Solution IV: Isopropanol: NOTE: Optional: RNase can be added to TE at final concentration of 20 g/ml. Procedure: 1. Inoculate a single bacterial colony from LB Agar medium to 100ml of LB broth with ampicillin. Incubate at 37C overnight. 2. Transfer the culture into an Eppendorf tube. Spin at 6K for 2min., decant the supernatant and repeat the step 2 twice. Drain the tube onto the paper towel. 3. Add 150l ice-cold Solution I to cell pellet and resuspend the cells as much as possible using micropipette. 4. Add 150l of Solution II, close the tubes and mix well by gently inverting the tubes for five times. 5. Add 150l ice-cold Solution III, close the tubes and mix the solution gently by inverting the tubes five times .Incubate the tubes on ice for 5 minutes. 6. Centrifuge the tubes at 12K for 10minutes. Transfer the supernatant to fresh microcentrifuge tube using clean disposable tip. Try to avoid taking any white precipitate during the transfer. 7. Add 450l of solution IV. Incubate the tube at room temperature for 2 minutes. 8. Centrifuge at 12K for 10 min at room temperature. Decant the supernatant. 9. To the pellet add 1 ml of ice-cold 70% ethanol. Close the tube and mix by inverting several times. Spin the tubes for 1 minute. Pour off the

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Cell and Molecular Biology Lab-06BTL47 supernatant (be careful not to dump out pellet) and drain tube on paper towel. 10. After drying the pellet, suspend in 100 l 1X TE. Observation: The amount of nucleic acid present in sample can be quantified using the absorbance using the absorbance at 260nm in a cuvette (quartz) using a spectrophotometer. An optical density is 1.0 is for 50g/ml for the double stranded DNA. The absorbance at 260/280 is 1.8 for pure DNA. The concentration can be determined by the following formula. Total nucleic acid (g) = [A260] [OD value] [Dilution factor] The molecular weight of the isolated plasmid can be determined on 1%agarose gel electrophoresis. Result: The concentration of the isolated plasmid DNA was found to be---------g/ml.

pUC 18 Plasmid

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Cell and Molecular Biology Lab-06BTL47

ISOLATION OF PLASMID DNA (SMALL SCALE)

Materials Required: Medium: LB broth Culture : 24hrs of culture E.coli Equipment: Orbital shaker, incubator and laminar hood. Others: Microfuge, micropipette, micropipette tips, ice bucket. Reagent: Solution I: STE buffer: 50 mM glucose 9 ml 50% glucose 25 mM Tris-HCl pH 8.012.5 ml 1 M Tris-HCl pH 8.0 10 ml 0.5 M EDTA pH 8.0 10 mM EDTA pH 8.0 RNase - 10mg/ml Make up the volume to 500 ml with distilled water Solution II: 10N NaOH/ 10% SDS 10% SDS - 100l 10 N NaOH - 20l Distilled water- 880l Total -1000l Solution III: 3M Potassium acetate: Solution IV: Isopropanol: RNase/TE: Optional: RNAse can be added to TE at final concentration of 20 ug/ml. Procedure: 1. Inoculate a single bacterial colony from LB Agar medium to 100ml of LB broth with 10l ampicillin. Incubate at 37C in orbital shaker overnight. 2. Transfer the culture into an Eppendorf tube. Spin at 6K for 10min, decant the supernatant and repeat the step 2 twice. Drain the tube onto the paper towel. 3. Add 100l of ice-cold Solution I to cell pellet and resuspend cells as much as possible using micropipette. 4. Add 200l of Solution II, (prepare fresh)close the tubes and mix well by gently inverting the tubes for five times. 5. Add 150l ice-cold Solution III, close the tubes and mix the solution gently by inverting the tubes five times .Incubate the tubes on ice for 5 minutes. 6. Centrifuge the tubes at 13K for 5minutes. Transfer the supernatant to fresh microcentrifuge tube using clean disposable tip. Try to avoid taking any white precipitate during the transfer. 7. Add equal volume of phenol :CIA. Mix by vortexing. 7. Add 500l of solution IV. Incubate the tube at room temperature for 2 minutes. 8. Centrifuge at 13K for 5 min at room temperature. Decant the supernatant. 9. To the pellet add 1 ml of ice-cold 70% ethanol. Close the tube and mix by inverting several times. Spin the tubes for 1 minute. Pour off the supernatant (be careful not to 19

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Cell and Molecular Biology Lab-06BTL47 dump out pellet) and drain the tube by inverting on paper towel to remove the excess ethanol. 10. After drying the pellet, suspend in 40 l 1X TE. Observation: The amount of nucleic acid present in sample can be quantified using the absorbance using the absorbance at 260nm in a cuvette (quartz) using a spectrophotometer. An optical density is 1.0 is for 50g/ml for the double stranded DNA. The absorbance at 260/280 is 1.8 for pure DNA. The concentration can be determined by the following formula. Total nucleic acid (g) = [A260] [OD value] [Dilution factor] The molecular weight of the isolated plasmid can be determined on 1%agarose gel electrophoresis. Result: The concentration of the isolated plasmid DNA was found to be---------g/ml.

ISOLATION OF GENOMIC DNA FROM E.Coli

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Cell and Molecular Biology Lab-06BTL47 Objective: To isolate the genomic DNA from E.coli cells. Introduction : Nucleic acids are the vital macromolecules in all living cell. The DNA contains the basic genetic information. The cellular DNA is located at the site of primary genetic activity (nucleus) within the cell. In prokaryotic cells, genetic activity occurs throughout the cell while in eukaryotic cells it lies in discrete particles within the cells Most of the DNA of eukaryotes lies in the nuclei and the remaining DNA in the partially self duplicating mitochondrial and chloroplast particles. The nuclear DNA combines with histone proteins in an orderly manner to form chromatin. The isolation of DNA from E.coli is a relatively simple process. The efficiency and recovery of extraction depends on the sample material, ionic conditions of the extraction medium, type of lysing agent used etc. Principle: The procedure for isolation of genomic DNA follows four stages: i) A culture of E.coli is grown and harvested. ii) The cells are lysed to release the contents. iii) The cell extract is treated to remove the other components except the DNA. iv) The resulting DNA is concentrated. The organism to be used should be grow in favorable medium at an optimal temperature and should be harvested in late log to early stationary phase for maximum yield. The lysis of the bacteria is initiated by resuspending the bacterial pellet in a buffer containing lysosyme and EDTA. The EDTA disrupts the outer membrane of the gram negative enevelope by removing the Mg2+ from the lipopolycaccharide layer and also inhibits DNAases. This allows the lysosyme access to the petidoglycan. After partial disruption of the peptidoglycan by the enzyme, a detergent such as SDS is added to lyse the cells. Most gram negative bacteria can be lysed without the lysosyme. Once the cells are lysed, the solution should be treated gently to prevent breakage of the DNA strands. The next step involves the separation of the DNA from other macromolecules in the lysate. The organic solvents like chloroform and isoamylalcohol is used to dissociate protein from nucleic acids. These reagents also remove lipids and polysacchrides. The supernatant is further treated with sodium acetate and isopropanol to precipitate the DNA Proteinase K and RNase treatment degrades the any further proteins and RNA if present in the lysate. Further the DNA is concentrated with ethanol. Materials Required: Medium: Luria bertani broth (LB) broth Tryptone - 10g Yeast extract - 5g Sodium chloride - 10g Distilled water -1000ml pH - 7.0-7.2 Culture : 24hrs of culture E.coli Reagent: TE buffer: 100ml 10 mM Tris-HCl pH 8.0 -1ml from 1 M Tris-HCl pH 8.0 Department of Biotechnology, CMRIT, Bangalore-37. 21

Cell and Molecular Biology Lab-06BTL47 1 mM EDTA pH 8.0 Distilled water -0.5 ml 0.5 M EDTA pH 8.0 -98.5 ml

1% SDS - 100ml SDS -1g Distilled water -100ml Heat gently to dissolve SDS. 5M Sodium chloride Sodium chloride 29.2g Distilled water 100ml Isopropanol: 70% Ethanol: NOTE: Optional: RNase can be added to TE at final concentration of 20 g/ml. Procedure: 1. Inoculate a single bacterial colony from LB Agar medium to 100ml of LB Broth and incubate at 37C overnight. 2. Transfer 1.5ml of culture into an Eppendorf tube. Spin at 5000rpm for 2min., decant the supernatant & repeat the step 2 twice. Drain the tube onto the paper towel. 3. Resuspend the pellet in 300 l of TE buffer. Incubate at room temperature for 20mins. 4. Add 1ml of 5M NaCl with SDS incubate in boiling water for 20 minutes. 5. After incubation centrifuge the tubes at 13000 rpm for 15minutes. 6. To the supernatant add chloroform: isoamylalcohol (24:1). Centrifuge the tubes at 13000rpm for 15minutes. 7. To the aqueous phase add 650l of isopropanol and centrifuge at 13000rpm for 15minutes. 8. Wash the pellet thrice with 70% ethanol. Dry the pellet. 9. Dissolve the pellet with 50l of TE buffer and store at -20C. 10. Run the sample on 0.8% of agarose gel. Observation: Ref. the previous experiment. Result: The concentration of the isolated genomic DNA was found to be---------g/ml.

ISOLATION OF P
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Department of Biotechnology, CMRIT, Bangalore-37.

Cell and Molecular Biology Lab-06BTL47

LANT DNA USING CTAB EXTRACTION METHOD

Objective: To isolate the plant DNA using CTAB extraction method. Introduction: Nucleic acids are the vital macromolecules in all living cell. The DNA contains the basic genetic information. The cellular DNA is located at the site of primary genetic activity (nucleus) within the cell. In prokaryotic cells, genetic activity occurs throughout the cell while in eukaryotic cells it lies in discrete particles within the cells Most of the DNA of eukaryotes lies in the nuclei and the remaining DNA in the partially self duplicating mitochondrial and chloroplast particles. The nuclear DNA combines with histone proteins in an orderly manner to form chromatin. Principle: DNA extraction from plant tissue can vary depending on the material used( leaf, stem, flowers, and roots. Generally young tissues with meristematic cells produce higher DNA yield, whereas certain highly differentiated tissues such as xylem, are poor source for DNA extraction. Each plant is different and it often contain special compounds such as poly phenols, mucilage -highly complex polysaccharides, pigments, secondary metabolites etc., which forms complexes with the DNA.CTAB(Cetyl trimethyl ammonium bromide) based extraction method are widely used for extraction of genomic DNA from a broad range of plant tissue. The first step being essentially mechanical means of breaking down the cell wall and membranes to allow the access to nuclear material.CTAB is nonanionic detergent which differentially solubilze the nucleic acid and polysaccharide under various salt conditions. When this solution is mixed with chloroform, the nonpolar compounds in the mixture dissolve into the chloroform layer, away from the aqueous DNA layer. Subsequently when weakly polar isopropanol is added to the aqueous solution, the largely negatively charged DNA molecules precipitate out as visible strands. Materials Required: Sample: Leaf of Reagent: Genomic DNA Lysis buffer: 100ml 10 mM Tris-HCl pH 8.0 -1ml from 1 M Tris-HCl pH 8.0 1 mM EDTA pH 8.0 -0.5 ml 0.5 M EDTA pH 8.0 Distilled water - 98.5 ml Genomic Suspension buffer - 100ml Genomic DNA Lysis buffer - 100ml Precipitation solution -10ml Agarose: RNase/TE: Optional: RNAse can be added to TE at final concentration of 20 g/ml. Procedure: 1. Take 100mg of the leaf sample crush it into powder in mortar and pestle. 2. Add 250l of Genomic DNA suspension buffer and crush until it form a fine paste.

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47 3. Pipette out this to 1.5ml of Eppendorf tube. Add 250l more of genomic DNA suspension buffer to rinse the mortar and pestle. Ensure that entire ground tissue material is transferred into the vial. 4. To the above add 5 l of the RNase solution provided. Mix 5-6 times by inverting the vial. Incubate at 65C for 10minutes with intermittent mixing. 5. To the above add 650 l Genomic lysis buffer and mix well. Incubate at 65C for 15minutes. Cool it to room temperature. 6. Centrifuge at 10,000rpm for 5minutes at room temperature. Collect the clear supernatant in a 1.5ml vial. 7. Decant 600l of the supernatant into two vials (each).Add 600l of precipitation solution to both the vials. Mix slowly by inverting the tubes until white strands of genomic DNA is seen separating out at interphase of two liquids. 8. Centrifuge at 10,000rpm for 15minutes and discard the supernatant. Wash the pellet with 70% ethanol. 9. Air-dry the pellet for 10minutes and then add 50l of 1X TE to both the vials. Incubate the solution at 50-55C for 10-15minutes or at 4C overnight. 10. Add 10l of 6X GLB to the solution and load 25l of the sample on a1% agarose gel along the along with 20l of the control genomic DNA. 11. Run the gel at 100V for 1hr. Visualize the DNA under UV light. Observe the mobility of the extracted DNA with that of the control DNA. Observation: The amount of nucleic acid present in sample can be quantified using the absorbance using the absorbance at 260nm in a cuvette (quartz) using a spectrophotometer . An optical density is 1.0 is for 50g/ml for the double stranded DNA. The absorbance at 260/280 is 1.8 for pure DNA. The concentration can be determined by the following formula. Total nucleic acid (g)= [A260 ][OD value] [Dilution factor] The molecular weight of the isolated plasmid can be determined on agarose gel electrophoresis. Result: The concentration of the isolated genomic DNA was found to be---------g/ml.

AGAROSE GEL ELECTROPHORESIS

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47 Objective: To separate DNA fragments by agarose gel electrophoresis. Introduction: Electrophoresis is a method to separate charged molecules under the influence of an electric field. DNA can be checked for size, homogeneity, and purity by this technique. This technique is simple, rapid and relatively an inexpensive method and capable of resolving DNA fragments that cannot be separated by any other methods. The velocity at which the molecules migrate depends on the size of the molecule, its molecular weight, conformation and the magnitude of net charge on the molecule. Agarose is a linear polysaccharide made up of the basic units of arabinose which comprises alternating units of galactose and 3, 6 anhydrogalactose. It is obtained from sea weeds and is reactive stable medium. Agarose gels have a lower resolving power but have a greater range of separation when compared to polysaccharide and polyacrylamide gels. DNA from 200bp to approximately 50kb in length, can be separated on agarose gel of various concentrations. A gel of 0.8% -1% is used for resolving fragments of size above 1Kb whereas 1.2-2% gel is used for separation of fragments below 1Kb. Principle: Agarose gels are invariably run as horizontal, submarine or submerged gels, so named because the gel is totally immersed in the buffer. Since DNA is polyanionic due to negative charge in the phosphate group. Hence, it moves towards the anode. Resolution is based on their molecular weight of the DNA fragment. It also depends on the buffer, gel matrix, molecular size of DNA, agarose concentration and voltage applied. The fragment with the low molecular weight moves fastest while the fragment with high molecular weight is found near the well. The DNA fragment separated can be visualized as fluorescent orange bands due to the staining of the DNA with ethidium bromide(which intercalates with RNA and DNA bases) when observed under an UV transilluminator. Since the distance of migration of DNA in the gel is inversely related to the log of molecular weight, the molecular weight of the sample can be known by using a standard molecular weight marker Materials Required: TrisMedium: Agarose Culture : 24hrs of culture E.coli Reagent: TAE buffer: 100ml 10 mM Tris-HCl pH 8.0- 1ml from 1 M Tris-HCl pH 8.0 1 mM EDTA pH 8.0 - 0.5 ml 0.5 M EDTA pH 8.0 Distilled water - 98.5 ml Staining dye: Ethidium bromide Procedure: Casting the 1% Agarose gel: 1. Prepare 1X TAE by diluting appropriate amount of 50X TAE with distilled water. 2. Weigh 0.5g of agarose and add to 50ml of 1X TAE. Boil the solution till the agarose dissolves completely and a clear solution is seen.

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47 3. Clean the gel casting tray and place it on horizontal platform. Place the combs of the electrophoresis set in the tank such that it is 2cm away from one end of the plate. Take care that the teeth of the comb should not touch the glass plate. 4. Pour the melted agarose solution into the tank without generating air bubbles. 5. Allow it to solidify at room temperature for an a hour. 6. Pour 1X TAE in the gel tank till the buffer level is seen to submerge the gel and the electrodes. 7. Gently lift the combs from the solidified agarose gel making sure that the wells are intact. 8. Add 3l of the staining dye to the buffer and pre-run the gel for about 10minutes after mixing the buffer and the staining dye. Electrophoresis: 1. Connect the power cord to the power supply according to convention. 2. Load the samples in the desired order and note down the order in which the samples are loaded. 3. Set the voltage to 100V and switch on the power supply. 4. Switch off when the bromophenol dye (tracking dye) runs till the other end of the gel. 5. Remove the gel from the tank using a gel scoop and visualize the DNA under UV- light. NOTE: Wear the gloves while handling all aspects of gel electrophoresis as ethidium bromide is mutagenic. Wear safety glasses for viewing the gel under UV light. OBSERVATION: Plasmid DNA may appear in one of five conformations on agarose gels during electrophoresis: Nicked open-circular DNA, linear DNA, Relaxed circular DNA, Super coiled DNA and super coiled denatured DNA.

Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, stained with ethidium bromide. The DNA size marker is a commercial 1 kbp ladder. The position of the wells and direction of DNA migration is noted.

PLASMID GENE MAPPING IN E. coli.

Department of Biotechnology, CMRIT, Bangalore-37. 26

Cell and Molecular Biology Lab-06BTL47

Objective: To study the gene mapping in the modified plasmid pUC 18 by using restriction endonucleases HindIII and EcoRI. Introduction: Digestion of the plasmid with any one of these endonucleases will makes a single cut in the plasmid, which linearizes the circular plasmid DNA and allows it to recombine with foreign DNA that has been cut with the same endonuclease. Principle: Two commonly used plasmid vectors for molecular biology techniques are pUC 18 and pUC 19. The modified pUC 18 consists of 5.5kb. P stands for plasmid and UC stands for University of California were it was first constructed. It includes a gene for antibiotic resistance to Ampicillin, and a lacZ gene for the enzyme betagalactosidase. The lacZ gene contains a polylinker for a series of unique restriction sites. This particular plasmid consists of three restriction sites for EcoRI, two sites for HindIII. Materials Required: Restriction Endonuclease(5U/l): HindIII, EcoRI Others:Assay buffer ,500bp ladder ,Agarose, 6x Gel loading dye, 50X TAE, Ethidium bromide Equipment: Agarose gel casting unit, power pack, micropipette and UV transilluminator. Procedure: 1. Prepare the three digestion mixture as follows: EcoRI reaction mixture Plasmid DNA 15l Assay buffer 3l EcoRI enzyme(5U/ l) 1l Water 11 l Total 30 l HindIII reaction mixture: Plasmid DNA 15l Assay buffer 3l HindIII enzyme(5U/ l) 1l Water 11 l Total 30 l EcoRI and HindIII reaction mixture: Plasmid DNA 15l Assay buffer 3l EcoRI enzyme(5U/ l) 1l Hindi (5U/ l) 1 l Water 10 l Total 30 l Procedure 27

Department of Biotechnology, CMRIT, Bangalore-37.

Cell and Molecular Biology Lab-06BTL47 1. Add all the components for the mix and tap it for 2-3times for mixing. Spin at 10000rpm for 20 Sec. 2. Incubate the mix at 37C in dry bath for 1hr. 3. Load the 30l of digested samples, 25l of undigested plasmid and 20 l of 500bp ladder on to 1% agarose gel. 4. Electrophorese the samples for 1hr at 100V. 5. Visualize the gel under UV transilluminator. Observation: Note down the size of the fragments as seen on Agarose gel under UV comparing with that of the ladder. Restriction endonuclease No. of sites No. of fragments EcoRI HindIII EcoRI + HindIII

Result: The digested plasmid DNA is separated by gel electrophoresis and the fragment sizes of pUC18 are mapped.

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47

RESTRICTION DIGESTION OF GENOMIC DNA

Objective: To digest genomic DNA of E.coli with EcoRI , BamHI and HindIII. Introduction: Restriction enzymes are bacterial enzymes that bind and cleave DNA at specific target site. It first discovered by Werner Arber . There are three types of restriction enzymes designated as I, II and III. Type II restriction enzymes are the most widely used in molecular biology applications. These enzymes bind DNA at specific recognition site, consisting of a short palindromic sequence, and cleave at within this site. Some enzymes cut both the DNA strand at the same position, creating blunt ended DNA fragments. Some enzymes create 5 overhangs and others create 3 overhangs Sticky ended fragments can be easily ligated to other sticky ended overhangs resulting in efficient cloning. Blunt ended fragments usually ligate less efficiently, making cloning more difficult. Restriction sites of the endonuclease Restriction Enzyme EcoRI Target site G AATT C C TTAAG HindIII
5'-A^A G C T T-3' 3'-T T C G A^A-5'

Overhangs Sticky ends

Blunt ends

BamHI

Blunt ends

Principle: This particular experiment demonstrates the function of restriction enzymes which cleaves DNA at specific sites. One unit of the enzyme is defined as the amount of the enzyme to completely digest 1g of substrate DNA at 37C under given conditions. One can find out the number of cleavage sites in a specific restriction enzyme by running a gel, after incubation. The molecular weight of the different DNA fragments can be determined by running a standard marker along with the samples. Materials Required: Restriction Endonuclease(5U/l): HindIII, EcoRI , BamHI Others: Assay buffer ,500bp ladder ,Agarose, 6x Gel loading dye, 50X TAE, Ethidium bromide Equipment: Agarose gel casting unit, power pack, micropipette and UV transilluminator. Procedure: 1. Prepare the three digestion mixture as follows: EcoRI reaction mixture Genomic DNA 15l 29

Department of Biotechnology, CMRIT, Bangalore-37.

Cell and Molecular Biology Lab-06BTL47 Assay buffer 3l EcoRI enzyme(5U/ l) 1l Water 11 l Total 30 l BamHI reaction mixture: Genomic DNA 15l Assay buffer 3l BamHI enzyme(5U/ l) 1l Water 11 l Total 30 l HindIII reaction mixture: Plasmid DNA 15l Assay buffer 3l HindIII enzyme(5U/ l) 1l Water 11 l Total 30 l 2. Add all the components for the mix and tap it for 2-3times for mixing. Spin at 10000rpm for 20 Sec. 3. Incubate the mix at 37C in dry bath for 1hr. 4. Load the 30l of digested samples, 25l of undigested plasmid and 20 l of 500bp ladder on to 0.8% agarose gel. 5. Electrophorese the samples for 1hr at 100V. 6. Visualize the gel under UV transilluminator. Observation: Note down the size of the fragments as seen on agarose gel under UV comparing with that of the ladder. Restriction endonuclease EcoRI BamHI HindIII No. of fragments No. of sites

Result: The size of the fragments is ____, ______ and ______ for EcoRI, BamHI and HindIII respectively.

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47

TRANSFORMATION OF E.coli CELLS

Objectives: 1. To prepare competent cells of E.coli. 2. To transform E.coli with pUC18. Introduction: Transformation is a phenomenon of introduction of exogenous DNA into conjugated cells, making the cell to acquire a new phenotype. It was first demonstrated in Streptococcus pneumoniae by Oswald Avery, Mac Leod and Mc Carty in 1944. It is widely used to transfer plasmid along with the target gene into the bacteria. Competent cells allow the DNA to enter the cell through the pores or channels in the cell membrane. Principle: Competence is the ability of a cell to take up extracellular DNA from its environment. Cells are made competent in the laboratory by treating with calcium chloride and subjecting to heat shock method. Here the competent cells are mixed with DNA is taken up during a heat shock step when the the cells are exposed briefly to a temperature of 42C. Immediately chilling on ice ensures closure of pores. Selection of cells containing transformed DNA is greatly enhanced by the antibiotic marker such as ampicillin . Materials Required: Medium:LB broth and Agar Luria Bertani (LB) broth Tryptone : 10g Yeast extract : 5g Sodiumchloride : 10g PH : 7.0-7.3 For Agar medium add Agar : 15g Culture: E.coli cultures (grown at 370C for 24 hrs). Plasmid: pUC18 Antibiotics: Ampicillin Equipment : Incubator, Orbital shaker, micropipettes Waterbath, spectrophotometer, Others: Conical flask, petriplates, pipettes and spreader.tips, crushed ice Procedure: Preparation of competent cells: Day 1 1. Under sterile condition break open the lyophilized vial. Streak the cell pellet onto LB plates in duplicates. 2. Incubate at 37C overnight. Day2 3. Inoculate a single colony into 5ml of LB medium and incubate for overnight at 37 C. Day3 Preparation of competent cells: 4. Inoculate 0.5ml of the overnight grown culture into 50ml LB media in a 250ml conical flask and incubate in a 37C Shaker. Grow until the OD A600 reaches 0.3. This takes 2-3hrs. 5. Chill the culture flask on ice for 20 minutes. 6. Transfer the culture under aseptic conditions into the sterile 2ml vials and spin Department of Biotechnology, CMRIT, Bangalore-37. 31

Cell and Molecular Biology Lab-06BTL47 at 3500rpm for 12minutes in a cooling centrifuge. 7. Discard the supernatant and add aseptically 500l of ice cold competent cell solution to each of the vials placed on the ice. Incubate the cells on ice for 20minutes. 8. Centrifuge the vials at 3500rpm for 12minutes at 4C. 9. Discard the supernatant and resuspend gently in 100l of ice cold competent cell solution. 10. Competent cells must be immediately transformed. Transformation of E.coli cells. Add 5l of the pUC18 into the aliquot of competent cells. Gently tap the cells after addition of the DNA and place the vial back on ice immediately. To the two vials of competent cells do not add the plasmid, this serves as control. Incubate all the vials on ice for 20minutes. Place the competent cells in 42C water bath for exactly 2minutes and quickly place on ice bath for 5minutes.This step is known as Heat shock method. Add 900l of LB broth under aseptic conditions to each of the vials and Incubate at 37C in Orbital shaker for 1hr.This is called recovery step. This allows the bacteria to express the antibiotic resistance gene. Add 100l of LB broth on the LB agar plates with ampicillin and then add 100l of the transformed cells on the plates. Mix well and spread thoroughly using a spreader. Plate 100l of untransformed cells on LB-Ampicillin plate to check for contamination. This serves as negative control. Plate 100l of untransformed cells on LB plate. This serves as positive control. Incubate all the plates at 37C overnight. Observe the colonies and calculate the transformation efficiency. Observation: Calculation of transformation efficiency as given: Amount of DNA transformed= 100ng Volume of the total culture = 1ml Volume of culture plated= 100l/ml Thus the amount of DNA plated = 10ng Transformation efficiency = No. of colonies X 1000 10 Result: The transformation efficiency was found to be ______ colonies/g

Department of Biotechnology, CMRIT, Bangalore-37.

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Cell and Molecular Biology Lab-06BTL47

Selection of recombinants in E.coli culture (Blue-white screening).

Objective: To isolate recombinants in E.coli cultures. Introduction: Selection of recombinants is very important process in biotechnology. The recombinant E.coli cells are isolated on medium containing marker (resistance to antibiotics) or reporter gene like LacZ. The lacZ gene codes for the enzyme called beta-galactosidase. Normally beta-galactosidase metabolizes lactose producing two products, galactose and glucose. X-Gal is a colorless sugar that can be metabolized by beta-galactosidase, producing galactose and a product that is blue. Therefore, X-Gal provides an easy way to determine whether the lacZ gene is functional. Materials: E.coli cells with recombinant and non recombinant plasmid pUC18, Lab plates, X gal solution, IPTG solution. Procedure: 1. Transfer aseptically 200 mL of the bacterial culture (transformed E.coli cells with recombinant plasmid pUC18) to an eppendorf tube. 2. Add 40 mL of Xgal solution and 8 mL of IPTG solution to the eppendorf tube containing the bacteria. Mix well by vortexing shortly. 3. Spread the mixed Xgal/IPTG-bacterial culture on a LB-agar plates for parent bacteria and on a LB-amp-agar plate for transformant bacteria. 4. Keep the spreaded plate at 37oC incubator for 12-16 hours. 5. If b-galactosidase gene is expressed, the blue colonies will form. If not, the white colonies may be observed for Escherichia coli.

Plate showing both blue and white colonies. Observation: Observe the blue and white colored colonies on LB plate. Result: White colored colonies having recombinant plasmid are isolated.

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Cell and Molecular Biology Lab-06BTL47

Tn 5 INDUCED MUTAGENESIS IN E.coli

Objective: To demonstrate Tn-5 mutagenesis by replica plating technique.. Introduction: Transposons are sequences of DNA with an ability to move from one site to another within the genome or to a plasmid. The insertion of the transposon usually abolishes the activity of agene. Some transposons carry drug resistance or other markers in addition to the function concerned with their transposition and are named as Tn followed by a number. Principle: The E.coli strain DH5 carries a transposon on its chromosomal DNA for Tn5. The transconjugant obtained by conjugation which is teracycline and streptomycin resistant. Any one of these genes might undergo insertional mutagenesis due to transposon. Thus the transconjugant becomes susceptible to the antibiotic. Thus insertional mutation induced by Tn5 can be monitored by allowing the individual colonies to grow on selective media. ( ie., with different antibiotics) The colonies that are incapable of growing in replica plate are supposed to be mutated by Tn-5. Materials Required: Medium: LB Agar plates containing Streptomycin and Tetracycline. LB Agar plates with Streptomycin LB Agar plates with Tetracycline. Luria Bertani (LB)Agar Tryptone Yeast extract Sodiumchloride Agar PH : : 10g : 5g : 10g : 15g 7.0-7.3

Culture: E.coli cultures (grown at 370C for 24 hrs): donor and recipient strain. Antibiotics: Streptomycin (100g/ml) and Tetracycline(30g/ml) Equipment : Incubator . Others : Sterile tooth picks. Procedure: 1. The plate with colonies that appeared on transconjugant is selected. 2. Prepare the master plate on the LB agar medium containing tetracycline and streptomycin. 3. Draw the lines on the reverse side of the petriplate and number as shown in the figure. Inoculate the transconjugant onto the master plate by streaking in each of the numbered box. Incubate at 37C for 24hrs.

1 2 3 4 5 6 Department of Biotechnology, CMRIT, Bangalore-37. 7 8 9 10 11 12 13

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Cell and Molecular Biology Lab-06BTL47

1 6

3 4

7 8

9 10 11 12

13 14 15 16 17 18 19 20 21 22 3 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

4. The replica of the master plate is picked up with the sterile replica block and pressed on to the LB medium with tetracycline and another replica on LB medium with Streptomycin. The third replica is pressed on to the LB medium with both tetracycline and streptomycin. 5. Incubate all the three plates at 37C for 24-48hrs. Observations: Make a note of the colonies that appear and those which did not appear on replica plates. .LBLB - Streptomycin Tetracycline+Streptomycin Not Appeared Appeared` Not Appeared Appeared` LB-Tetracycline Not Appeared Appeared

Result: In tetracycline plate, numbered colony and in streptomycin plate did not grow and indiccting that the gene responsible for antibiotic resistance was non functional due to insertion of Tn 5 transposon .

STUDY OF BACTERIAL CONJUGATION

Department of Biotechnology, CMRIT, Bangalore-37. 35

Cell and Molecular Biology Lab-06BTL47 Objective: To demonstrate Plasmid mediated conjugation between F+ donor and Frecipient E.coli . Introduction: Conjugation is the process of exchange of genetic material between two bacterial cells through cell to cell contact. It was first discovered by Lederberg and Tatum in 1946. It is a mechanism of horizontal gene transfer, which may be plasmid or chromosomal DNA mediated. Conjugation involves two bacterial strains, one donor cell and other recipient cell. The donor cell contains a fertility plasmid or F plasmid that contains genes, which control the synthesis of sex pili and of enzymes that induce DNA exchange. Principle: The Kit consists of two bacterial strains, donor strain and the recipient strain. The donor stain carries a tetracycline resistance plasmid and recipient strain carries streptomycin resistance plasmid. Both the organism is grown on their respective antibiotic selection media and both the strains are incubated for the conjugation process. The conjugated sample is plated on the media containing both the antibiotics. Only the conjugated sample survives on media containing both tetracycline and streptomycin indicating that gene transfer has taken place. Materials Required: Medium: Luria Bertani (LB)broth Tryptone : 10g Yeast extract : 5g Sodiumchloride : 10g PH : 7.0-7.3 Culture: 24hrs of culture E.coli culture donor and recipient strain Antibioptics: Streptomycin (100g/ml) and Tetracycline(30g/ml) Equipment : Orbital Shaker, Procedure: Day 1 1. Under sterile condition break open the lyophilized vial, add 400l LB broth and incubate for one hour with shaking (160rpm) in the incubator at 37C. Streak or spread 200 l of the donor strain on LB medium containing tetracycline (30g/ml) and recipient strain on LB plate containing streptomycin (100g/ml) and incubate for overnight at 37C. Day2 2. Inoculate a single colony from the donor and the recipient plate into 5ml of LB broth containing respective antibiotics. 3. Incubate at 37C in Orbital shaker. Day 3 4. To 25ml LB broth add 1ml of overnight grown cultures of donor and recipient stains with their respective antibiotics( Donor : Tetracycline : Recipient : streptomycin). Incubate at 37C in Orbital shaker. 5. Grow the strains until the OD of both the stains reaches 0.8-0.9 at absorbance 600nm. 6. Take three sterile test tubes. Label them as Donor, Recipient and conjugation mix. 7. Transfer 0.2ml culture from donor and recipient into the respective tubes without the anti biotic. Department of Biotechnology, CMRIT, Bangalore-37. 36

Cell and Molecular Biology Lab-06BTL47 8. To the conjugation tube, add equal volume of each of the cultures and swirl the tube. Incubate all the tubes in 37C incubator for 1.5hrs.of the ( No shaking should be done during incubation) 9. After incubation add 2ml of sterile LB into each tube and continue the incubation for 1.5hrs. at 37C. 10. After 1.5hrs, remove the tubes from the incubator. Spread 100l of each sapmle on to three types of plates ie., LB with tetracycline (30g/ml),LB with Streptomycin (100g/ml) and LB with both the antibiotics (Tetracycline and Streptomycin). Incubate all the plates in an inverted position at 37C overnight. Observation: Tabulate the observations in the following pattern Plate Donor strain Recepient Strain Conjugated Result: The conjugation has occurred in E.coli cells. LB-Tetracycline LB - Streptomycin LB-Tetracycline + Streptomycin

Diagrammatic representation of bacterial conjugation

SODIUM DODECYL SUPLHATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) OF PROTEINS

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Department of Biotechnology, CMRIT, Bangalore-37.

Cell and Molecular Biology Lab-06BTL47

Objective: To separate proteins by using SDS-PAGE. Introduction: Electrophoresis is widely used to separate and characterize proteins by applying electric current. Electrophoretic procedures are rapid and relatively sensitive requiring only micro-weights of proteins; electrophoresis in gel is more convent than any other medium such as paper and starch gel. Electrophoresis of proteins in polyacrylamide gels is carried out in buffer gels (non-denaturing) as well in sodium dodecyl sulphate (SDS) containing (denaturing) gels. Separation in buffer gel relies on the both charge and size of protein whereas it depends only upon the size in the SDS-gels. Analysis and comparison of proteins in a large number of samples is easily made on polyacrylamide gel slabs. Polyacrylamide gels are formed by polymerizing acrylamide with cross linking agent (bisacrylamide) in the presence of a catalyst (persulphate ion) and chain initiator (TEMED; N,N,N N tetramethylethylene diamine). Solutions are normally degassed by evacuation prior to polymerization since oxygen inhibits polymerization. The porosity of gel is determined by the relative proportion of acrylamide monomer to bisacrylamide. Gels are usually referred to in terms of total percentage of acrylamide and bisacrylamide, present and most protein separations are performed using gels in the range 7-15%. A low percentage gel is used to separate high molecular weight proteins and vice-versa. At high concentration of persulphate and TEMED the rate of polymerization is also high. Among a number of methods commonly used the sodium dodecyl sulphate-polyacryalamide gel electrophoresis (SDS-PAGE) in slabs are described below.

Picture of a SDS-PAGE. The molecular marker is in the left lane

Principle
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Department of Biotechnology, CMRIT, Bangalore-37.

Cell and Molecular Biology Lab-06BTL47 SDS is an anionic detergent which binds strongly to and denatures proteins. The number of SDS molecules bound to polypeptide chain is approximately half the number of amino acid residues in that chain. The protein SDS complex carries net negative changes, hence move towards the anode and the separation is based in size of the protein.

Note: To separate many different protein molecules of different shapes and sizes, first denature the proteins with SDS so that the proteins no longer have any secondary, tertiary or quaternary structure (i.e. retain only their primary amino acid structure). Consider two proteins that are each 500 amino acids long but one is shaped like a closed umbrella while the other one looks like an open umbrella. SDS is denaturing all proteins to the same linear shape. Materials Required: Stock Acrylamide Solution Acrylamide 30% Bisacrylamide 0.8% 0.8 g Water to 100ml Separating Gel buffer 1.875M Tris- HCl 22.7 g Water to 100ml pH 8.8 Stacking gel buffer 0.6 M Tris-HCl 7.26g Water to 100ml pH 6.8 Polymerizing agents a. Ammonium persulphate 5% 0.5 g 10 ml prepare freshly before use b. TEMED fresh from refrigerator. Department of Biotechnology, CMRIT, Bangalore-37. 39

Cell and Molecular Biology Lab-06BTL47 Electrode buffer 0.05 M Tris 12g 0.192M Glycine 28.8g 0.1% SDS 2g Water to 2 L pH 8.2 to 8.4 (No adjustment required) (may be used 2-3 times) Sample buffer (5X concentrations) Tris HCl buffer pH 5ml SDS 0.5g Sucrose 5g Mercaptoethanol 0.25ml Bromophenol blue (0.5% W/V solution in water) 1 ml Water to 10 ml Store in frozen aliquots. Dilute to 1x concentration and use. Sodium dodecyl sulphate 10% solution-store at room temperature. Standard marker proteins Protein stain solution Coomassie brilliant blue R 250 0.1g Methanol 40ml Acetic acid 10ml Water 50ml First dissolve the dye in the methanol and proceed. Use fresh preparations every time. Destainer- As Above without the dye.

Procedure 1. Thoroughly clean and dry the glass plates and spacers, then assemble them properly. Hold the assembly together with bulldog clips. Clamp in an upright position. White petroleum jelly or 2% agar (melted in a boiling water bath) is then applied around the edges of the glass plates.

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Cell and Molecular Biology Lab-06BTL47 2. Prepare sufficient volume of separating gel mixture (30 ml for a chamber of about (18*9*0.1 cm) by mixing following. For 15% gel Stock acrylamide solution Tris HCl (pH8.8) Water 10%SDS TEMED 20 ml 8ml 11.4ml 0.4ml 20microL for 10%gel 13.3 ml 8ml 18.1ml 0.2ml 0.4ml 20microL

Ammonium persulphate solution 0.2ml

3. Mix gently and carefully, pour the gel solution in the chamber between the glass plates. Layer distilled water on top of the gel and leave to set for 30-60min. 4. Prepare stacking gel (4%) by mixing the following solutions (total volume 10ml) Stock acrylamide solution = 1.35 ml Tris HCl (pH6.8) = 1ml Water = 7.5 ml Degas the above, and then add: Ammonium persulphate (5%) = 50 micro L 10%SDS = 0.1ml TEMED = 10 micro L Remove the water from the top of the gel and wash with a little stacking gel mixture, place the comb in the stacking gel and allow the gel to set (30-60min) 5. After the stacking gel has polymerized, remove the comb without disporting the shapes of the well. Carefully install the gel after removing the clips, agar etc, in the electrophoresis apparatus. Fill it with electrode buffer and remove any trapped air bubbles at the bottom of the gel. Connect the cathode at the top and turn on the DC-power briefly to check the electrode circuit. The electrode buffer and the plates can be kept cooled using a suitable facility so that heat generated during the run is dissipated and does not affect the gel and resolution.

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Cell and Molecular Biology Lab-06BTL47 6. Prepare the sample solutions and take up the required volume in a micro syringe and carefully inject into a sample using the 5strength sample buffer and water in such a way that the sample amount of protein is present in unit volume. Again the concentration should be such as to give a sufficient amount of protein (50-200microL)in a volume (25-30 microL) not greater than the size of the sample well. As general practice, heat sample solutions in boiling water for 2-3 min to ensure complete interaction between the proteins and SDS. 7. Cool the sample solutions and take up the required volume in a micro syringe and carefully inject it into a sample well through the electrode buffer. Making the position of wells on the glass plate with marker pen and the presence of Bromophenol blue in the sample buffer facilitate easy loading of the samples. Similarly load a few wells with standard marker proteins in the sample buffer. 8. Turn on the current to 10-15mA for initial 10-15 min until the samples travel through stacking gel. The stacking gel helps concentration of the samples. The continue the run at 30mA until the Bromophenol blue reach the bottom of the gel. However, the gel may be run at a high current (60-70mA) for short period (1hr) with proper cooling. 9. After the run is complete, carefully remove the gel from between the plates and immerse in staining solution for at least 3h or overnight with uniform shaking. The proteins absorb Coomassie brilliant blue. 10. Transfer the gel to a suitable container with at least 200-300ml distaining solution and shake gently and continuously. Dye that is not bound to proteins is thus removed. Change the desatiner frequently, particularly during initial periods, until the background of the gel is colorless. The proteins fractionated into bands are see colored blue. As the proteins of minute quantities are stained faintly, distaining process should be stopped at appropriate stage to visualize as many bands as possible. The gel can now be

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Cell and Molecular Biology Lab-06BTL47 photographed or stored in a polyethylene bags or dried for permanent record. Observation: Observe and compare the protein bands in SDS-PAGE

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Cell and Molecular Biology Lab-06BTL47

POLYMERASE CHAIN REACTION

Objective: To amplify the given DNA fragment by PCR. Introduction: Polymerase chain reaction (PCR) was first demonstrated by Kary Mullis in 1984 for which he won Nobel Prize. This method involves invitro amplification of DNA. Specific DNA fragments can be amplified using specific primers. The advantage of PCR is that it can be used to amplify picograms of DNA and it can detect one or more DNA copies in a heterogeneous sample, thereby allowing convenient screening of a single transformant. This technique finds greater applications to amplify the cloned insert DNA in research, to identify polymorphism among genotypes in plant breeding programmes, to identify the pathogens on seed materials, to identify the biological parents, culprits in crimes such as murder, etc. in forensic diagnosis. The DNA samples for PCR may be from few hr-old embryos or from tissue thousands of years after death. Principle: DNA amplification is a very simple method for invitro amplification of specific nucleic acids using Taq DNA polymerase and minimum two oligonucleotides specific to DNA to be amplified. Earlier E.coli DNA polymerase was used in PCR; but since it is heat sensitive fresh enzyme was added after each cycle. Taq DNA polymerase has been isolated from bacterium Thermus aquaticus which lives in water at 75C. Its DNA polymerase has a temperature optimum at 72C and is reasonably stable even at 94C. Hence it is not required to be added after each cycle. The PCR technique involves three basic steps for DNA amplification. 1) Denaturation of DNA into single strands at 94C to 98 C. 2) Annealing of primer at 37C -65C to each original strand for new strand synthesis. 3) Extension of the new DNA strands from the primer. Materials Required: Taq DNA polymerase: 3 units / l 10 X Taq DNA polymerse Buffer: 10mMDeoxynucleotide triphosphate(dNTPs): 2.5mM concentration of each dNTP and 10mM of final mix. DNA Template: Genomic DNA From __________, with 200ng/ l. Primers: These are 18-28 bases long oligonucleotides with melting temperature of 54C, with 250ng/l. Equipment: Thermal cycler, Agarose, electrophoresis unit, UV transilluminator. Others: Eppendorf vials, micropipette ethidium bromide, agarose gel, paraffin oil Procedure: 1. Prepare DNA amplification mix in l (reagents in ice- bucket) as follows: 10X Taq polymerase buffer 5l 2mM d NTP 3l Template 1l Forward Primer (0.5g/ml) 1l Reverse primer (0.5g/ml) 1l Taq DNA polymerase 1l Department of Biotechnology, CMRIT, Bangalore-37. 44

Cell and Molecular Biology Lab-06BTL47 Sterile double distilled water 38l Total volume 50l 2. Transfer 10l aliquots of this mix into 0.5ml microfuge tubes and mix the solution gently. 3. Set up the control tube with all the reagents except the enzyme. 4. Overlay each of the tubes with 50 l of light liquid paraffin or mineral oil to prevent evaporation during the process. 5. Amplification was carried out using the following reaction condition for at least 30 cycles. Initial denaturation -94C 1minute Denaturation -94C - 1minute Annealing - 48C 30 seconds Ex tension - 72C 1minute Final extension - 72C 2minutes.

6. Amplification was carried out in a thermal cycler. 7. After the reaction, 10 l of the reaction mixture in the aqueous layer was run on 1% agarose gel for 1-2hrs at 100V. Observation: The amplified DNA was subjected to electrophoresis to confirm the success of PCR amplification using a standard molecular weight for comparing. Result: Amplified band corresponding to __Kb marker was found.

Diagrammatic representation of PCR

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