mol bio manual

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 auto-

dispenser 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.

Department of Biotechnology, CMRIT, Bangalore-37. 1


CONTENTS

Sl.No Name of the Experiment Page No

1 Study of divisional stages in Mitosis. 3-5

2 Study of divisional stages in Meiosis. 6-8

3 Study of Polytene and Lampbrush chromosomes. 9-10

4 Isolation of plant protoplasts by enzymatic method. 11-13

5 Chemical fusion of plant protoplasts (PEG, Calcium). 14-15

6 Isolation of plasmid DNA from E.coli. 16-20

7 Isolation of genomic DNA (plant/ animal/ microbial sources). 21-24

8 Agarose gel electrophoresis and quantification of nucleic acids.

(colorimetric, ethidium bromide dot blot and standard DNA marker)

25-26

9 Plasmid gene mapping in E.coli 27-28

10 Restriction mapping/ digestion of genomic DNA. 29-30

11 Transformation of E.coli cells. 31-32

12 Selection of recombinants in E.coli (Blue-white screening). 33

13 Tns 5 induced mutagenesis in E.coli. 34-35

14 Study of conjugation in E.coli. 36-37

15 SDS-PAGE. 38-43

16 PCR (Demo experiment). 44-45



STUDY OF DIVISIONAL STAGES IN MITOSIS

Objective: To study the different stages in mitosis

Introduction: 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.

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


http://www.accessexcellence.org/RC/VL/GG/human.html


Objective: To observe different stages of mitosis in the onion root tip cells.

Materials required: Onion root tipsEquipments: Waterbath, Microscopes, Spirit lamp Reagents: 45% Acetic acid.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 materialsRoot 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 1h5. 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 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

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 Metaphase 1: Homologous chromosomes align at the equatorial plate. o Anaphase 1: Homologous pairs separate with sister chromatids

remaining together. o Telophase 1: Two daughter cells are formed with each daughter

containing only one chromosome of the homologous pair. Second division of meiosis: Gamete formation

o Prophase 2: DNA does not replicate. o Metaphase 2: Chromosomes align at the equatorial plate. o Anaphase 2: Centromeres divide and sister chromatids migrate

separately to each pole. o Telophase 2: Cell division is complete. Four haploid daughter cells are

obtained.

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.

Materials required: Onion root tips


http://www.accessexcellence.org/RC/VL/GG/comparison.html

http://www.accessexcellence.org/RC/VL/GG/comeiosis.html


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 aceto-

carmine. 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:



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,


http://en.wikipedia.org/wiki/Amphibians

http://en.wikipedia.org/wiki/Oocyte

http://www.answers.com/topic/insect

http://www.answers.com/topic/malpighian-tubule-system

http://www.answers.com/topic/chironomidae-1

http://www.answers.com/topic/salivary-gland

http://www.answers.com/topic/gene-expression-2

http://www.answers.com/topic/transcription

http://www.answers.com/topic/chromosome

http://www.answers.com/topic/mitosis

http://www.answers.com/topic/cell-division

http://www.answers.com/topic/dna-replication

http://www.answers.com/topic/dna-replication


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.



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.



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 Amount

A Mineral Salts

1 Ammonium Nitrate 82.5 g/l

2 Potassium Nitrate 95.0 g/l

3 Boric acid 1190 mg/l

4 Potassium Dihydrogen Phosphate 34 mg/l

5 Potassium Iodide 166 mg/l

6 Sodium Molybdate 250 mg/l

7 Cobalt Chloride Dihydrate 25.0 mg/l

8 Calcium Chloride Dihydrate 88.0 gm/l

9 Magnesium Sulphate Heptahydrate 74.0 gm/l

10 Manganese Sulphate tetrahydrate 4460 mg/l

11 Zinc Sulphate Heptahydrate 1720 mg/l

12 Copper Sulphate Pentahydrate 25.0 mg/l



13 Ethylene Diamino Tetra Acetic acid Disodium salt 7.45 g/l

14 Ferrous Sulphate heptahydrate 5.57 g/l

B Sugars

1 Glucose 30.0 g/l

2 Ribose 0.5 g/l

3 Mannose 0.5 g/l

4 Sorbitol 0.5 g/l

C Organic Acids

1 Sodium Pyruvate 5.0 mg/l

2 Citric acid 10.0 mg/l

3 Malic acid 10.0 mg/l

D Vitamins

1 Thiamine Hydrochloride 100 mg/l

2 Nicotinamide 500 mg/l

3 Pyridoxine Hydrochloride 500 mg/l

4 Calcium pantothenate 250 mg/l

5 Biotin 100 mg/l

6 Ascorbic acid 75.0 mg/l

7 Riboflavin 50.0 mg/l

8 Inositol 100 gm/l

E Hormones

1 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) 0.5 mg/l



2 Benzyl Amino Purine (BAP) 1.0 mg/l

Observation: Observe the isolated protoplasts

Pre-digestion Post-digestion Protoplasts

CHEMICAL FUSION OF PLANT PROTOPLASTS (PEG, CALCIUM)

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.


http://www.biology.lsu.edu/webfac/nkato/methods/methodImages/PreDigestedLeaves.jpg

http://www.biology.lsu.edu/webfac/nkato/methods/methodImages/PostDigestedLeaves.jpg

http://www.biology.lsu.edu/webfac/nkato/methods/methodImages/Protoplast.jpg


2. After centrifugation, the tubes are placed in a water bath (370c) for 40-50 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 micro quantities ●.

1. 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. 2-3 drops of PEG are added from periphery in each Petri plate which is Incubated 30min at room temperature. 3. After PEG treatment, protoplasts are gradually washed in medium

( during this process most of the fusion is achieved). 4. 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.



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



precipitation can therefore purify DNA from protein contaminants. Ethanol helps to remove the remaining salts and SDS from the preparation.

Materials Required:Medium: LB brothCulture: 24hrs of culture E.coliReagent:Solution I: GTE buffer: 50 mM glucose -9 ml 50% glucose25 mM Tris-HCl pH 8.0 -12.5 ml 1 M Tris-HCl pH 8.0

10 mM EDTA pH 8.0 -10 ml 0.5 M EDTA pH 8.0

RNase -100µg/mlMake up the volume to 500 ml with distilled water

Solution II: 0.2N NaOH/ 1% SDS1% SDS -50 ml 10% SDS0.2 N NaOH -100 ml 1 N NaOHMake 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 µg/ml.

Procedure:1. Inoculate a single bacterial colony from LB Agar medium to 100ml of

LB broth with ampicillin. Incubate at 37°C 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 150µl ice-cold Solution I to cell pellet and resuspend the cells as much as possible using micropipette.

4. Add 150µl of Solution II, close the tubes and mix well by gently inverting the tubes for five times.

5. Add 150µl 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 450µl 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



supernatant (be careful not to dump out pellet) and drain tube on paper towel.

10. After drying the pellet, suspend in 100 µl 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 50µg/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 (µg) = [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----------µg/ml.

pUC 18 Plasmid



ISOLATION OF PLASMID DNA (SMALL SCALE)

Materials Required:

Medium: LB brothCulture : 24hrs of culture E.coliEquipment: Orbital shaker, incubator and laminar hood.Others: Microfuge, micropipette, micropipette tips, ice bucket.Reagent:Solution I: STE buffer: 50 mM glucose - 9 ml 50% glucose25 mM Tris-HCl pH 8.0- 12.5 ml 1 M Tris-HCl pH 8.0

10 mM EDTA pH 8.0 10 ml 0.5 M EDTA pH 8.0

RNase - 10mg/mlMake up the volume to 500 ml with distilled water

Solution II: 10N NaOH/ 10% SDS10% SDS - 100µl10 N NaOH - 20µlDistilled water- 880µlTotal -1000µlSolution 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 10µl ampicillin. Incubate at 37°C 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 100µl of ice-cold Solution I to cell pellet and resuspend cells as much as possible using micropipette. 4. Add 200µl of Solution II, (prepare fresh)close the tubes and mix well by gently inverting the tubes for five times. 5. Add 150µl 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 500µl 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



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 µl 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 50µg/ml for the double stranded DNA. The absorbance at 260/280 is 1.8 for pure DNA.


Total nucleic acid (µg) = [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----------µg/ml.

ISOLATION OF GENOMIC DNA FROM E.Coli



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 - 7.0-7.2Culture : 24hrs of culture E.coli

Reagent:TE buffer: 100ml10 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

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 µg/ml.

Procedure:1. Inoculate a single bacterial colony from LB Agar medium to 100ml of

LB Broth and incubate at 37°C 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 µl 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 650µl of isopropanol and centrifuge at

13000rpm for 15minutes.8. Wash the pellet thrice with 70% ethanol. Dry the pellet.9. Dissolve the pellet with 50µl of TE buffer and store at -20°C.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---------- µg/ml.

ISOLATION OF P



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: 100ml10 mM Tris-HCl pH 8.0 -1ml from 1 M Tris-HCl pH 8.01 mM EDTA pH 8.0 -0.5 ml 0.5 M EDTA pH 8.0 Distilled water - 98.5 mlGenomic Suspension buffer - 100ml Genomic DNA Lysis buffer - 100mlPrecipitation solution -10mlAgarose:

RNase/TE: Optional: RNAse can be added to TE at final concentration of 20 µg/ml.

Procedure:

1. Take 100mg of the leaf sample crush it into powder in mortar and pestle.

2. Add 250µl of Genomic DNA suspension buffer and crush until it form a fine paste.



3. Pipette out this to 1.5ml of Eppendorf tube. Add 250µl 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 µl of the RNase solution provided. Mix 5-6 times by inverting the vial. Incubate at 65°C for 10minutes with intermittent mixing.

5. To the above add 650 µl Genomic lysis buffer and mix well. Incubate at 65°C 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 600µl of the supernatant into two vials (each).Add 600µl 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 50µl of 1X TE to both the vials. Incubate the solution at 50-55°C for 10-15minutes or at 4°C overnight.

10. Add 10µl of 6X GLB to the solution and load 25µl of the sample on a1% agarose gel along the along with 20µl 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 50µg/ml for the double stranded DNA. The absorbance at 260/280 is 1.8 for pure DNA.


Total nucleic acid (µg)= [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---------- µg/ml.

AGAROSE GEL ELECTROPHORESIS



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: Tris-Medium: AgaroseCulture : 24hrs of culture E.coliReagent:TAE buffer: 100ml

10 mM Tris-HCl pH 8.0- 1ml from 1 M Tris-HCl pH 8.01 mM EDTA pH 8.0 - 0.5 ml 0.5 M EDTA pH 8.0Distilled water - 98.5 ml

Staining dye: Ethidium bromideProcedure: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.



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 3µl 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.



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 beta-galactosidase. 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/µl): HindIII, EcoRI Others:Assay buffer ,500bp ladder ,Agarose, 6x Gel loading dye, 50X TAE, Ethidium bromideEquipment: Agarose gel casting unit, power pack, micropipette and UV transilluminator.

Procedure:1. Prepare the three digestion mixture as follows:EcoRI reaction mixturePlasmid DNA 15µlAssay buffer 3µlEcoRI enzyme(5U/ µl) 1µlWater 11 µlTotal 30 µl

HindIII reaction mixture:Plasmid DNA 15µlAssay buffer 3µlHindIII enzyme(5U/ µl) 1µlWater 11 µlTotal 30 µl

EcoRI and HindIII reaction mixture:Plasmid DNA 15µlAssay buffer 3µlEcoRI enzyme(5U/ µl) 1µlHindi (5U/ µl) 1 µlWater 10 µlTotal 30 µl

Procedure



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 37°C in dry bath for 1hr.3. Load the 30µl of digested samples, 25µl of undigested plasmid and 20 µl 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

EcoRI HindIII EcoRI + HindIII

No. of sitesNo. of fragments

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



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 Target site Overhangs

EcoRI G AATT C

C TTAAG

Sticky ends

HindIII 5'-A^A G C T T-3' 3'-T T C G A^A-5'

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 1µg of substrate DNA at 37°C 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/µl): HindIII, EcoRI , BamHI Others: Assay buffer ,500bp ladder ,Agarose, 6x Gel loading dye, 50X TAE,

Ethidium bromideEquipment: Agarose gel casting unit, power pack, micropipette and UV

transilluminator.

Procedure:1. Prepare the three digestion mixture as follows:EcoRI reaction mixtureGenomic DNA 15µl



Assay buffer 3µlEcoRI enzyme(5U/ µl) 1µlWater 11 µlTotal 30 µl

BamHI reaction mixture:Genomic DNA 15µlAssay buffer 3µlBamHI enzyme(5U/ µl) 1µlWater 11 µlTotal 30 µl

HindIII reaction mixture:Plasmid DNA 15µlAssay buffer 3µlHindIII enzyme(5U/ µl) 1µlWater 11 µlTotal 30 µl

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 37°C in dry bath for 1hr.4. Load the 30µl of digested samples, 25µl of undigested plasmid and 20 µl 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 HindIIINo. of fragmentsNo. of sites

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



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 42°C. 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 : 15gCulture: E.coli cultures (grown at 370C for 24 hrs). Plasmid: pUC18Antibiotics: AmpicillinEquipment : 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 37°C 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 37°C 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



at 3500rpm for 12minutes in a cooling centrifuge. 7. Discard the supernatant and add aseptically 500µl 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 4°C.9. Discard the supernatant and resuspend gently in 100µl of ice cold competent cell solution.10. Competent cells must be immediately transformed. Transformation of E.coli cells.

Add 5µl 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 42°C water bath for exactly 2minutes and quickly place on ice bath for 5minutes.This step is known as Heat shock method.

Add 900µl of LB broth under aseptic conditions to each of the vials and Incubate at 37°C in Orbital shaker for 1hr.This is called recovery step. This allows the bacteria to express the antibiotic resistance gene.

Add 100µl of LB broth on the LB agar plates with ampicillin and then add 100µl of the transformed cells on the plates. Mix well and spread thoroughly using a spreader.

Plate 100µl of untransformed cells on LB-Ampicillin plate to check for contamination. This serves as negative control.

Plate 100µl of untransformed cells on LB plate. This serves as positive control.

Incubate all the plates at 37°C 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= 100µl/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/µg



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.

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 DH5α 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 : 10g Yeast extract : 5g Sodiumchloride : 10g Agar : 15g PH : 7.0-7.3

Culture: E.coli cultures (grown at 370C for 24 hrs): donor and recipient strain.Antibiotics: Streptomycin (100µg/ml) and Tetracycline(30µg/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 37°C for 24hrs.


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


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 37°C for 24-48hrs.

Observations:

Make a note of the colonies that appear and those which did not appear on replica plates.

.LB-Tetracycline+Streptomycin

LB - Streptomycin LB-Tetracycline

Not Appeared Appeared` Not Appeared Appeared` 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 .


1 2 3 4 5 6 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


STUDY OF BACTERIAL CONJUGATION

Objective: To demonstrate Plasmid mediated conjugation between F+ donor and F-

recipient 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 : 7.0-7.3Culture: 24hrs of culture E.coli culture donor and recipient strainAntibioptics: Streptomycin (100µg/ml) and Tetracycline(30µg/ml)

Equipment : Orbital Shaker,

Procedure:Day 11. Under sterile condition break open the lyophilized vial, add 400µl LB broth and incubate for one hour with shaking (160rpm) in the incubator at 37°C. Streak or spread 200 µl of the donor strain on LB medium containing tetracycline (30µg/ml) and recipient strain on LB plate containing streptomycin (100µg/ml) and incubate for overnight at 37°C.Day22. Inoculate a single colony from the donor and the recipient plate into 5ml of LB broth containing respective antibiotics.3. Incubate at 37°C in Orbital shaker.

Day 34. 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 37°C 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.8. To the conjugation tube, add equal volume of each of the cultures and swirl the tube. Incubate all the tubes in 37°C 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 37°C.10. After 1.5hrs, remove the tubes from the incubator. Spread 100µl of each sapmle on to three types of plates ie., LB with tetracycline (30µg/ml),LB with Streptomycin (100µg/ml) and LB with both the antibiotics (Tetracycline and Streptomycin). Incubate all the plates in an inverted position at 37°C overnight.

Observation: Tabulate the observations in the following pattern

Plate LB-Tetracycline LB - Streptomycin LB-Tetracycline + Streptomycin

Donor strain Recepient StrainConjugated

Result: The conjugation has occurred in E.coli cells.

Diagrammatic representation of bacterial conjugation



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

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


http://en.wikipedia.org/wiki/Image:SDS-PAGE.jpg


Principle

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.

- 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.

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 for 10%gel

Stock acrylamide solution 20 ml 13.3 ml

Tris –HCl (pH8.8) 8ml 8ml

Water 11.4ml 18.1ml

Ammonium persulphate solution 0.2ml 0.2ml

10%SDS 0.4ml 0.4ml

TEMED 20microL 20microL

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.

6. Prepare the sample solutions and take up the required volume in a

micro syringe and carefully inject into a sample using the 5-

strength 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

photographed or stored in a polyethylene bags or dried for

permanent record.

Observation: Observe and compare the protein bands in SDS-PAGE



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 75°C. Its DNA polymerase has a temperature optimum at 72°C and is reasonably stable even at 94°C. 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 94°C to 98° C. 2) Annealing of primer at 37°C -65°C 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 / µl 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/ µl. Primers: These are 18-28 bases long oligonucleotides with melting temperature of 54°C, with 250ng/µl.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 µl (reagents in ice- bucket) as follows: 10X Taq polymerase buffer 5µl 2mM d NTP 3µl Template 1µl Forward Primer (0.5µg/ml) 1µl Reverse primer (0.5µg/ml) 1µl Taq DNA polymerase 1µl



Sterile double distilled water 38µl Total volume 50µl 2. Transfer 10µl 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 µl 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 -94°C – 1minute Denaturation -94°C - 1minute Annealing - 48°C – 30 seconds Ex tension - 72°C – 1minute

Final extension - 72°C – 2minutes.

6. Amplification was carried out in a thermal cycler.7. After the reaction, 10 µl 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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