INTRODUCTIONBiotechnologyBiotechnology is a field of applied biology that involves the use of living things in engineering, technology, medicine, and other useful applications. Modern use similar term includes genetic engineering as well as cell- and tissue culture technologies. The concept encompasses a wide range of procedures for modifying living organisms according to human purposes - going back to domestication of animals, cultivation of plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. By comparison to biotechnology, bioengineering is generally thought of as a related field with its emphasis more on higher systems approaches (not necessarily altering or using biological materials directly) for interfacing with and utilizing living things.The United Nations Convention on Biological Diversity defines biotechnology as:"Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."Biotechnology draws on the pure biological sciences (genetics, microbiology, animal cell culture, molecular biology, biochemistry, embryology, cell biology) and in many instances is also dependent on knowledge and methods from outside the sphere of biology (chemical engineering, bioprocess engineering, information technology, and biorobotics). Conversely, modern biological sciences (including even concepts such as molecular ecology) are intimately entwined and dependent on the methods developed through biotechnology and what is commonly thought of as the life sciences industry.Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non food (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses.For example, one application of biotechnology is the directed use of organisms for the manufacture of organic products (examples include beer and milk products). Another example is using naturally present bacteria by the mining industry in bioleaching. Biotechnology is also used to recycle, treat waste, clean up sites contaminated by industrial activities (bioremediation), and also to produce biological weapons.Molecular biology is the study of biology at a molecular level. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated. The term molecular biology was coined in 1938 by Warren Weaver, then director of the natural sciences program at the Rockefeller Foundation.Techniques of Molecular BiologySince the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.Expression cloningOne of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection.In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signalling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.Polymerase chain reaction (PCR)The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.Macromolecule blotting and probingThe terms northern, western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting, after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the northern blot actually didn't use the term. Further combinations of these techniques produced such terms as southwestern (protein-DNA hybridizations), northwestern (to detect protein-RNA interactions) and farwestern (protein-protein interactions), all of which are presently found in the literature.Southern blottingNamed after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels; however non-radioactive alternatives are now available. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines.Western blottingAntibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies) or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including coloured products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, these immunostaining methods, such as FISH, are used more often in cell biology research.Eastern blottingEastern blotting technique is to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.MATERIALS AND METHODSDNA Isolation from BacteriaDNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional step in a DNA extraction:Breaking the cells open commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample.Removing membrane lipids by adding a detergent.Removing proteins by adding a protease (optional but almost always done).Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+. This stops dnase enzymes from degrading the DNA.Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.Reagents required:TE buffer: 10 mM Tris-Cl (pH, usually 7.6 or 8.0), 1 mM EDTA (pH 8.0) (To make it take 1 ml of 1M Tris (pH 8.0), 0.2 ml of 0.5 M EDTA (pH 8.0) and add water to make 100 ml)10% (w/v) sodium dodecyl sulfatephenol\chloroform (50:50)Isopropanol70% ethanol3M sodium acetate pH 5.2: To prepare a 3 M solution: Dissolve 40.83 g of sodium acetate-3H2O in 80 ml of H2O. Adjust the pH to 5.2 with glacial acetic acid. Adjust the volume to 100 ml with H2O. Dispense into aliquots and sterilize by autoclaving.20 mg/ml proteinase KProtocol:Grow E. coli culture overnight in rich Luria broth. Transfer 2 ml of culture to a 2-ml micro centrifuge tube and spin for 2 min at 5000 rpm. Decant the supernatant and drain well onto a tissue Paper.Re-suspend the pellet in 467 μl TE buffer by repeated pipetting. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K, mix, and incubate 1 hr at 37°C. Add an equal volume of phenol/chloroform and mix well but very gently to avoid shearing the DNA by inverting the tube until the phases are completely mixed. CAUTION: Phenol causes severe burns, wear gloves goggles, and lab coat and keep tubes capped tightly. Spin at 12,000 rpm for 10 min.Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Mix by inversion for 4-5 times. Spin 10 min at 12,000 rpm.Transfer the upper aqueous phase to a new tube.Add 1/10 volume of sodium acetate. Mix by gentle inversion.Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end). Wash DNA by 1 ml of 70% ethanol for 30 sec.Repeat step 14, remove ethanol and dry DNA at room temperature (or at 37°C). Re-suspend DNA in at least 200 μl of TE buffer. Complete re-suspension may take several hours. Store DNA at 4°C for short term, and -20 or -80°C for long term. After DNA has dissolved, determine the concentration by measuring the absorbance at 260 nm.CTAB Method / Protocol for DNA Extraction from Plant Samples Reagents Required: CTAB buffer -2% CTAB (20gm CTAB) -20mM EDTA [40ml EDTA stock (0.5M)] -100mM Tris-Cl pH 8.0 [100ml Tris-Cl stock (1M)] -1.4M NaCl [280ml NaCl stock (5M)] -make up to 1 Litre with water, pH 7.5 - 8.0, and autoclave + 0.2% Mercaptoethanol Wash Buffer -70% Ethanol PVPPChlorophorm/isoamyl alcohol (24:1)Liquid NitrogenDNA Isolation ProtocolPreheat 5ml CTAB buffer (add 10µl Mercaptoethanol to each 5ml CTAB) in a blue-topped 50ml centrifuge tube at 60-65oC. Remove and discard midribs, and wrap leaves in aluminium foil and freeze in liquid nitrogen. 0.5–1.0 gm tissue/5ml CTAB (Can store leaf material after liquid Nitrogen for 1-2 days at –20 oC or –80 oC for longer periods) Gently crumble leaf tissue (1 gm) over cold pestle of liquid nitrogen. Grind frozen leaf with liquid nitrogen and add 0.5 spatula of PVPP powder after grinding. Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid leaving dry material around rim of tube. Adjust CTAB volume to give a slurry-like consistency, mix occasionally and incubate for 60 min at 60oC After this add equal volume of chloroform/iso-amyl alcohol (24:1), mix for about 3 min, and then transfer contents to centrifuge tubes. Balance by adding extra chloroform/iso-amyl. Spin 12,000rpm for 15min (ensure correct tubes used), brake off. (For extra pure DNA isolation - spin and retain supernatant before chloroform extraction). Remove supernatant with wide-bore pastette (cut off blue tip) to clean tube, repeat chloroform extraction once. Supernatant should be clear, though sometime may be colored. Precipitate DNA with 0.66 vol. of cold isopropanol, keep for 15 min at 4oC (can leave overnight). Spool out or spin down DNA, 2 min at 3,000rpm. Or add 2.5 volumes of ethanol and 0.1 volume sodium acetate and keep at RT overnight (will give better pracipitation).Add 1 ml wash buffer (70% ethanol) for 5 min and centrifuge for 2 min at 5000 rpm, discard supernatant. Repeat step 8.Dry pellet briefly and re-suspend in 150 ul TE buffer. (can be left overnight) Add 1µl 10mg/ml RNAse to each 1ml TE/DNA mixture and incubate for 60min at 37oC. (If RNase in the sample doesn't matter – stages 11 and 12 may be omitted) Repeat step 5 to 9.Spool DNA out. Air dry and resuspend in 50 to 100 ul TE or water (takes time) and freeze until required.Protocol Hints: The Phenol: Chloroform phase may separate with the plant debris, so it may be seen as two layers: debris and an organic phase. The sample may be left at room temperature for several hours to overnight without problems.You can leave the sample at this step for at least two days without problems. Although it still contains RNA, DNA taken at this stage of purification is generally suitable for restriction digestion. For plants containing high polysaccharide levels, i.e. glutinous sap, increase the concentration of CTAB to 3% or higher. For plants with high concentrations of phenolic compounds (i.e. oaks, walnuts) add 1% (w/v) polycinylpyrollidine (PVP-40). CAUTION! This Phenol is a biohazard!!!!!!! Plasmid IsolationThe DNA of prokaryotic cells (bacteria) is relatively simple in comparison to the DNA of eukaryotic cells. A bacterium has about 1/1000 as much DNA as a eukaryotic cell. In addition to chromosomal DNA, a bacterium may also carry an additional circular piece of DNA called a plasmid. The plasmid has proven to be a useful tool for the molecular biologist. The Mini-Prep procedure is used to isolate plasmid DNA from bacteria while limiting contamination from proteins and genomic DNA. Buffers and reagents: P1 solution: Resuspension Buffer (To be stored at 4oC)50 mM Tris HCl (pH 8.0)10 mM EDTA100 ug/ml RNAse AP2 Solution: Lysis Buffer (Room temp.)200mM NaOH, 1% SDSP3 Solution: Neutralization Buffer (Room Temp or 4oC)3 M Potassium Acetate, pH 5.5 Phenol (TE saturated) Chloroform: isoamylalcohol (24:1) Isopropanol 70% Ethanol Preparation of Buffers:P1: Dissolve 6.06 g Tris Base, 3.72 g EDTA.2H2O in 800 ml H2OAdjust pH to 8.0 with HClAdjust volume to 1 L with water. Autoclave and store at 4 oC.P2: Dissolve 8.0gm NaOH pellet in 950 ml H2O, 50 ml 20% SDS. The final volume should be 1 L.P3: Dissolve 294.5 Pottassium acetate in 500ml H2O, Adjust pH 5.5 with glacial acetic acid.Adjust the volume to 1 L with water.Or if we have stocks ready- P1: Tris HCl pH 8.8 (1M stock): 5mlEDTA (0.5 M Stock) : 2ml RNAse add separately (100ug/ml) Total is 7ml, now make it 100ml and store.P2: NaOH (1M Stock): 20 mlSDS (10%) : 10 ml, Make it 100 ml with water.P3: 3M Potassium acetate (pH 5.5)TE Buffer to dissolve/store the DNA.Tris HCl (pH 8.0): 10 mM EDTA (pH 8.0) : 1 mM So from stocks- For 10 ml = Tris HCl (pH 8.0) (1M) = 100 ul EDTA (pH 8.0) (0.5 M) = 20 ul H2O= 9.8 mlIn our experiment plasmid DNA will be isolated by the alkaline lysis method as follows-Bacterial cells were grown overnight (16 hrs.) in tubes containing 5 ml LB with appropriate antibiotics at 37oC. Pour ~1.4 ml of the culture into labeled 1.5 mL centrifuge tubes. Cells were pulsed down in microcentrifuge at high speed (14,000 rpm) for 30 seconds.Supernatant was discarded, and cells resuspended in 100µL of ice cold 100 l TE buffer (10 mM Tris - HCl pH 8.0, 1 mM EDTA, and pH 8.0) and 5l of RNase (20mg/ml). Mixed the content of the tube thoroughly, and then incubated in ice for 5 minutes. Added freshly prepared 200l lysis buffer (0.2N NaOH, 1% SDS), mixed by gently inverting and rolling the tube to make sure all the bacterial slurry along the sides and cap are mixed with lysis buffer, incubated on ice until translucent (for 5 min). Then added 150 l ice cold 3M potassium acetate, (pH 5.5), mixed by vortexing and mixture was kept in ice for 10 min. Micro-centrifuged at 12,000-14,000 rpm for 10 minutes, supernatant was removed to new microcentrifuge tubes. The white precipitate containing genomic DNA and proteins was pelleted down with this step. For phase separation the supernatant was carefully withdrawn and mixed by inversion with equal volume of phenol: chloroform: isoamyl alcohol mixture (25:24:1). The mixture was centrifuged at 12000 g for 10 min and the upper aqueous layer was taken.It was mixed with equal volume of chloroform: isoamyl alcohol (24:1) mixed and centrifuged at 12000 g for 10 min and the upper aqueous layer was taken. For precipitation of Plasmid DNA Isopropanol (0.6 V of supernatant) was added to supernatant and incubated for 10 min at 25C. The precipitated DNA was pelleted at 12,000 rpm for 15 min. Added ~1ml of 70% EtOH to wash pellet, gently mix by inverting. It was re-centrifuged for 5 minutes at 12,000-14,000 x g. Supernatant was discarded and plasmid DNA was vacuum dried. Plasmid DNA was dissolved in 50 ul TE buffer and stored at 4oC.The plasmid DNA was electrophoresed and analyzed with 0.7 % Agarose gel. RNA Isolation using Bio-Red RNA isolation kitRNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNAMaterial Required: Overnight grown bacterial culture. RNA Isolation Kit (Bio-Red)RNAse inhibitor (DEPC)70% Isopropanol (Prepare yourself)DEPC treated eppendorf and tips (1 ml and 200 ul)500 ug/ml Lysozyme in TEβ-MercaptoethanolWater BathBacteriaFollow steps 1-4, and then continue with step 1 of “Bacteria, and Yeast”B1. Transfer up to the equivalent of 3 OD/ml bacterial cultures into a 2 ml capped microcentrifuge tube (provided). Centrifuge at maximum speed for 1 min. Decant the supernatant, and blot the tube with paper towels.B2. Add 100 µl of 500 µg/ml lysozyme in TE (10 mM Tris, 1 mM EDTA, pH 7.5) to each tube, and pipet up and down to resuspend the pellet thoroughly. Incubate at room temperature for 5 min.B3. Add 350 µl of lysis solution (already supplemented with 1% β-mercapto-ethanol) to each tube, and pipet up and down at least 12 times to mix thoroughly.B4. Add 250 µl of 70% isopropanol (not supplied) to each tube, and pipet up and down to mix thoroughly. Make sure that no bilayer is visible, and that the viscosity is substantially reduced.YeastFollow steps C1–C6, and then continue with step 1 of “Cultured Cells, Bacteria,And Yeast (cont.)”C1. Prepare lyticase dilution buffer:1 M sorbitol0.1 M EDTA, pH 7.40.1% (v/v) β -MercaptoethanolEquilibrate the buffer at 30°C before use.C2. Transfer up to the equivalent of 3 OD/ml yeast culture into a 2 ml capped microcentrifuge tube (provided). Centrifuge at maximum speed for 1 min. Decant the supernatant, and blot the tube with paper towels.C3. Add 1 ml of 50 units/ml lyticase in lyticase dilution buffer equilibrated to 30°C, to each tube. Pipet up and down to resuspend the yeast pellet completely. Incubate for 10 min.C4. Centrifuge the tube at 5,000 rpm for 5 min. Decant the supernatant andGently blot the tube on paper towels.C5. Add 350 µl of lysis solution (already supplemented with 1% β-mercapto-ethanol) to each tube, and pipet up and down at least 12 times to mix thoroughly.C6. Add 350 µl of 70% ethanol (not supplied) to each tube, and pipet up and down to mix thoroughly. Make sure that no bilayer is visible, and that the viscosity is substantially reduced.Procedure: 1. At this time place the elution solution into a 70°C water bath to warm it for step 10. Insert an RNA binding column into a 2 ml capless wash tube (provided).2. Decant or pipet the homogenized lysate into the RNA binding column. Centrifuge for 30 sec. Remove the RNA binding column from the wash tube, discard the filtrate from the wash tube, and replace the column into the same wash tube.3. The low stringency wash solution is provided as a 5x concentrate. Add 4 volumes of 95–100% ethanol to the low stringency wash solution concentrate before initial use. This corresponds to 80 ml for the mini kit.4. Add 700 µl of low stringency wash solution to the RNA binding column. Centrifuge for 30 sec. Discard the low stringency wash solution from the wash tube, and replace the column into the same wash tube. 5. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not provided) to the vial and pipetting up and down briefly to mix. 6. For each column processed, mix 5 µl of reconstituted DNase I with 75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube (not provided). Scale up proportionally if processing more than one column. Add 80 µl of diluted DNase I to the membrane stack at the bottom of each column. Allow the digest to incubate at room temperature for 15 min. When the digest is complete, centrifuge the columns for 30 sec. Discard the digest buffer from the wash tube, and replace the column into the same wash tube.7. Add 700 µl of high stringency wash solution to the RNA binding column. Centrifuge for 30 sec. Discard the high stringency wash solution from the wash tube, and replace the column into the same wash tube. 8. Add 700 µl of low stringency wash solution to the RNA binding column. Centrifuge for 1 min. Discard the low stringency wash solution from the wash tube, and replace the column into the same wash tube. 9. Centrifuge for an additional 2 min to remove residual wash solution. Note: The elution solution should be 70°C before proceeding with the elution step.10. Transfer the RNA binding column to a 1.5 ml capped microcentrifuge tube (provided). Pipet 80 µl of the warmed elution solution onto the membrane stack at the bottom of the RNA binding column, and allow 1 min for the solution to saturate the membranes. Centrifuge for 2 min to elute the total RNA. The eluted total RNA samples can be used immediately in RT-PCR reactions or in any other application. Alternatively, the total RNA can be stored at 4°C for later use. Protein Isolation From PlantPolyacylamide Gel Electrophoresis (SDS-PAGE)Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. Support Matrices: Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation. Separation of Proteins and Nucleic AcidsProteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules. Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size. Principles of Gel Electrophoresis: Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. The gel itself is composed of either agarose or polyacrylamide, each of which has attributes suitable to particular tasks: Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.SDS- PAGE OF PROTEINSSeparation of Proteins under Denaturing conditionsSodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - i.e.: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- Mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. Determination of Molecular WeightThis is done by SDS-PAGE of proteins - or PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and it’s Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel. Continuous and Discontinuous Buffer SystemsThere are two types of buffer systems in electrophoresis, continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system (read about this in any textbook). ProtocolNB: Acrylamide monomer is a potent cumulative neurotoxin. do not mouth pipette acrylamide solutions, and wear gloves when handling unpolymerised solutions.Assembling gel apparatus:Assemble two glass plates (one notched) with two side spacers, clamps, grease, etc. as shown by demonstrators. Stand assembly upright using clamps as supports, on glass plate. Pour some pre-heated 1% agarose onto glass plate, place assembly in pool of agarose: this seals the bottom of the assembly. Resolving Gels:Gel concentration of 12.5% in 0.25 M Tris-HCl pH 8.8 ________________________________________Volume Volume Reagent:(ml: TO MAKE 30 ML)(ml: TO MAKE 10 ML)40% Acrylamide stock*: 9.43.1Water (distilled)12.33.81 M Tris-HCl pH 8.8 7.52.510% SDS 0.30.1Peroxydisulphate 1% 0.50.5TEMED (added last) 20 ul20 ul* = 19:1 - 38:1 w: w ratio of acrylamide to N, N’-methylene bis-acrylamide Mix ingredients GENTLY! In the order shown above, ensuring no air bubbles form. Pour into glass plate assembly CAREFULLY. Overlay gel with isopropanol to ensure a flat surface and to exclude air. Wash off isopropanol with water after gel has set (+15 min). Stacking Gels:Gel concentration of 4.5% in 0.125 M Tris-HCl pH 6.8 ________________________________________Volume Volume Reagent:(ml TO MAKE 15 ML)(ml TO MAKE 10 ML)40% Acrylamide stock 1.7 1.1Water 10.87.11 M Tris-HCl pH 6.8 1.91.2510% SDS 0.150.1Peroxydisulphate 1% 0.50.5TEMED (stir quickly)20 ul20 ul________________________________________Mix as before, then pour onto top of set resolving gel, insert comb, allow to set, remove comb, and fill with electrophoresis buffer. Assemble top tank onto glass plate assembly. Fill with electrophoresis buffer. Electrophoresis bufferThe final TANK buffer composition is 196 mM glycine / 0.1% SDS / 50mM Tris-HCl pH 8.3, made by diluting a 10x stock solution. This goes in both top and bottom tanks. Sample Preparation:1. Grind a little leaf material (eg. 2 grams) in a mortar. Centrifuge in an Eppendorf tube for 3 min. Take supernatant and mix 100ul 1:1 (v:v) with SDS-PAGE disruption mix: this is 125mM Tris-HCl pH 6.8 / 10% 2-mercaptoethanol / 10% SDS / 10% glycerol, containing a little bromophenol blue. BE CAREFUL WITH THIS AS IT SMELLS AWFUL and is poisonous to boot!! 2. For liquid / purified samples, take eg. 100 ul and add 50 - 100 ul of disruption mix. Centrifuge the above solution for 20 min at 15,000 rpm at 4 oC. Take supernatant and use directly for electrophoresis.3. Heat sample Eppendorfs for 5 min at 95oC in a "float" in a waterbath. Layer samples under buffer on stacking gels. Connect up apparatus and electrophorese as shown. Staining of Gels:Coomassie Brilliant Blue staining:Make up stain: 0.2% CBB in 45:45:10 % methanol: water: acetic acid. Cover gel with staining solution, seal in plastic box and leave overnight on shaker (RT) or for 2 to 3 hours at 37oC also with agitation. De-stain with 25:65:10 methanol: water: acetic acid mix, with agitation. Southern BlottingLocalization of particular sequences within genomic DNA is usually accomplished by the transfer techniques described by Southern (1975). Genomic DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually nitrocellulose filter or nylon membrane). The relative positions of the DNA fragments are preserved during their transfer to the filter. The DNA attached to the filter is hybridized to radiolabeled DNA or RNA, and autoradiography is used to locate the positions of bands complementary to the probe.Steps in Southern Blotting:1. Depurination of DNA fragments: Wash gel in 0.25 M HCl 5' (small gels), 7' (large gels).2. Denaturation: Wash gel in (0.5 M NaOH, 1.5 M NaCl) for 20' (small) to 30' (large).3. Neutralization: Wash in (0.5 M Tris pH 7.0, 3.0 M NaCl) for 20' (small) to 30' (large).4. Cut nylon membrane (MSI 0.45 micron #N04HY00010) and several pieces of blotting (e.g. Schleicher and Schuell GB002) paper to the same size as the gel. Wet the nylon with dH20, then soak in 5x SSC.5. Assemble sandwich:Large sheet of plastic wrap.Two pieces blot paper (precut to same size as gel) soaked in 20x SSC Gel (wells-side down)Presoaked nylonOne piece blot paper soaked in 5x SSC10-15 pieces of dry blot paperh. Wrap whole sandwich in the plastic wrapPlace glass plate and weight on top and let transfer >3 hr.6. Crosslink after blotting.Principle: To hybridize radioactively labeled DNA probes to Southern blots containing human DNA to detect Restriction Fragment Length Polymorphosims (RFLPs). This method can also be used for most other applications (e.g., slot blots, zoo blots, yeast YAC blots etc.). Time required: 2 days for hybridization, 3-5 days for X-ray film exposureProcedure: Day 1 Pre-hybridization: Rinse blots in Postwash II solution (0.1X SSC, 0.2%SDS). Place a piece of nylon mesh between every two blots and place the blots in a thermal sealing plastic blot bag. [At least 20 blots (20cm x 20cm each) can be hybridized in a single bag]. Add 25 ml of pre-hyb solution for 1-2 blots; increase volume for more blots (or enough to allow blots to slide slightly inside; use ~150ml for 20 blots). Double seal the bags completely with T-bar sealer and incubate at 50 degrees C for at least 30 minutes before adding the labeled probe. Leave enough open space in the bags to cut open and add probe. (You can also let the pre-hybridization incubation go overnight).Hybridization: The following steps should be carried out behind a plexiglass shield :Check the radioactive work area with the monitor before starting to work. If the probe is known to be "single copy" do not add competitor human DNA. Otherwise add 250 µl of sonicated human DNA (2.5 mg/ml stock) to the labeled probe DNA, place the tubes in the metal holder and denature DNA by boiling both the probe DNA mix and the labeled lambda DNA (used to reveal molecular weight standards) for 10 minutes. If the probe does not have human DNA added, 5 minutes of boiling is sufficient to denature DNA. Quick-cool the tubes in ice for ~2 minutes with the tubes still in the metal holder to prevent lids from popping open. Add the probe DNA and lambda DNA to ~3ml of pre-hyb solution in a 5 or 15 ml snap-cap tube, cap tightly and mix by inverting the tube. (Refer to Primer Extension labeling protocol for counts to be added). Cut open the pre-hyb bag at one corner, pour the probe and pre-hyb mix into the bag and reseal the bag (double sealing is good). Check for leaks by pushing the liquid towards the seals. Pass the bag over a sharp edge (e.g., the edge of the table) to help squeeze the hybridization mix evenly between the blots (the more blots in the bag, the more vigorous the mixing should be). Incubate the bags at 50 degrees C on the platform shaker in the radioactive-labeled incubator, overnight (16-24 hours). Clean up the radioactive work area thoroughly after you are done. Refer to the 32P lab safety sheet.Day 2 Post-hybridization washes to remove non-specifically bound probe: Steps 1 thru 3 below should be done behind the plexiglass shield and contaminated gloves should be changed frequently Cut open one corner of the bag behind the shield and pour the radioactive hyb mix carefully into the liquid radioactive waste container. If you are working with several bags at once pull the blots out first, drain the hyb mix into a large beaker and then transfer the liquid waste to the liquid waste container. Remove the blots from the bag and place in approximately 200 ml (or more according to the number of blots/bags, enough to immerse blots) of Postwash I solution [2X SSC (for 1 liter, mix 100 ml of 20X SSC with 900 ml of dH2O)] in a deep sided steel pan. The first wash solution should be disposed of in the liquid waste container. [The other washes can be poured down the sink marked for radioactive work. Record the amount of waste poured down the sink (a 25ng reaction is approximately equal to 0.05 µCi)]. Transfer the blots to a fresh 200 ml (or more) of Postwash I solution. Rub the blots individually at each wash, flip them over and rub again. Wash at room temperature in 2X SSC for a total of 30 minutes.The following steps can be done outside the radioactive work bench. Check the wash solution with the monitor and use your judgement. Wash the blots twice for 30-45 minutes each wash at 65 degrees C in Postwash II solution [ 0.1x SSC, 0.2% SDS (for 500 ml, add 2.5ml 20X SSC and 10 ml 10% SDS to 487.5ml dH2O)] in a covered steel pan with gentle shaking. These washes should be done in the 65 degrees C shaker-baths meant for radioactive work only. X-ray film exposures: Take the blots out of the shaker-bath and press the blots between two pieces of Whatman 3MM paper to remove excess wash solution. Check the Whatman papers with a monitor; if radioactivity is detected, wash blots one more time. DO NOT ALLOW THE BLOTS TO BECOME COMPLETELY DRY. Place the semi-dry blots in blot bags (14" x 17") as follows: cut open the bag on one side so that two contiguous sides are open and lightly crumple the bags with your hands to remove static between the sheets of the blot bags. This will help in slipping the blots easily between the plastic sheets of the bags. Smooth the bags to get good contact between the blot and the bag. Place blots as close together as possible without overlap and make sure they are placed asymmetrically in the bag for orientation purposes. Try to accommodate as many blots as possible per bag. Open the film holder (cardboard Kodak X-ray film exposure holder); place the blot bag inside, position the intensifying screen on top of the blot bag with the dull side of the screen in contact with blot bag. If multiple holders are used, orient all with the same side up as only one side of the holder has a lead lining. In the darkroom (with the safelight ON) open the Kodak X-ray film box, remove a sheet of film (the film feels stiff between two sheets of paper) and place film between the intensifying screen and the blot bag. Close the cassette holder and close the clasp. The X-ray film box is kept in the cabinets in the processor room. It should always be stored in the dark, with the lid securely taped. Place the film holder/s between the plexiglass or metal holder plates and store the holders in the -80 degrees C freezer for 3-5 days. The exposure time varies with the probes and the counts obtained. Prepare the film labels (printed labels); store them until the films are developed. Remove the film holder/s from the freezer and thaw at room temperature for 1 to 2 hours before processing the films. This is to prevent the screens from cracking (if bent), due to the intense cold. Develop the films using the Kodak X-ray film processor. Refer to the processor instructions written by Matt Holt in this manual. Place the labels on the developed films; mark the individuals of the family or pedigree appropriately with Sharpee markers (Other markers may not be removable with ethanol). Mark the alleles also, if they are known. If the film is overexposed, place another film in the holder and re-expose without the screen for 1 day. If the film is underexposed, re-expose with the intensifying screen for several days (up to 10 days maximum).Stripping blots for reuse: Remove blots (should be semi-moist) from the bags and place them in a steel pan containing appropriate amount of 0.1N NaOH. (For 500ml, add 5ml 10N NaOH to 495 ml dH2O). Shake the blots at room temperature for 30 minutes (no longer than 45 minutes) on a platform shaker. This removes the probe from the blot. Drain the wash. Neutralize the blots with 0.2M Tris-HCl pH 8.0, 0.1X SSC, 0.5% SDS (for 500 ml, add 100 ml of 1M Tris-HCl pH8.0, 2.5 ml 20X SSC and 25 ml 10% SDS to 372.5 ml of dH2O). Shake the blots at room temperature for 30 minutes or more on a platform shaker. Store the blots wet in sealed blot bags at room temperature or in the cold (4 degrees C) until further use. If the blots are in the general lab supply, sign them back into the record books, and file them in the appropriate places.Solutions: Tris-mix for 1 literfinal concentration500 ml 1M Tris-HCl, pH 7.50.5 M Tris-HCl50 ml 0.5M EDTA, pH 8.025 mM EDTA50 ml 50 mg/ml Heparin2.5 mg/ml Heparin150 ml 31/3 % Sodiumpyrophosphate0.5% Sodium pyrophosphate250 ml 10% Sarkosyl2.5% SarkosylPre-hyb solution for 1 literfinal concentration200 ml Tris-mix* (see above)0.1M Tris-HCl200 ml 50% Dextran sulfate10% Dextran sulfate200 ml 5M NaCl1M NaCl300 ml Formamide30% Formamide100 ml ddH2OStore at 4 degrees C. 50% Dextran sulfate: For 200 ml: Add 100 g dextran sulfate to 130 ml of ddH2O in a 500 ml bottle, let it sit overnight to dissolve and store at room temperature. Wear a face mask while weighing out dextran sulfate. Heparin (50mg/ml): Dissolve 5 g heparin in 50 ml of ddH2O, adjust volume to 100 ml with ddH2O, filter sterilize and store at 4 degrees C. 10% Sarkosyl: Dissolve 100 g N-lauroylsarcosine in 500 ml ddH2O and adjust volume to 1000 ml with dd2O. Store at room temperature. 5M Sodium chloride (NaCl): Dissolve 1168.8 g NaCl in 3000 ml of ddH2O, adjust volume to 4000ml with ddH2O in a 4 L carboy and store at room temperature. 10% SDS: Dissolve 100 g sodium dodecyl sulfate (SDS) in 500 ml of ddH2O, adjust volume to 1000 ml and store at room temperature. Wear a face mask while weighing out SDS. 20X SSC (20 liters): Dissolve 3504 g NaCl and 1760 g sodium citrate in 10 L ddH2O. (Use a 20 L carboy.) Adjust the volume to 20 L with ddH2O, and the pH to 7.4 with several drops of concentrated HCl. Competent Cell PreparationMaterials Required: 2X LB -500 ul. 25% PEG 3350 -400 ul.1 M Magnesium Chloride (MgCl2) -50 ul. DMSO (dimethylesulphoxide) -50 ul.This will give you 1 ml of TSS. You need 2 ml to resuspend pellet from 50ml culture. Prepare 2X Lb and autoclave them before making competent cells. Add all the four components accordingly and filter sterilize the TSS solution. Every time you have to make fresh TSS.During sterilization u will not get back the exact volume you have put through filter .So filter about 3 ml of TSS then only you will have enough 2ml TSS. Use the filter of 0.2 mm size this protocol will make competent cells in 10-20 min and having a high efficiency. Procedure: Grow an over night culture.In morning add 1 % of inoculum to 50ml medium (i.e. 500 ul of overnight culture)Grow the 50ml culture till 0.3-0.4 OD at 37°C.Then spin down the cell at 4°C at 3000-4000 rpm for 5 min.Mix 2 ml of chilled TSS solution to cell pallet and mix gently with pipette.Aliquot 100 ul in 1.5ml eppendorfs, freeze in liquid nitrogen and store at -70°C.You can use these cells for 6 months.TransformationTransformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA). This is a common laboratory technique in molecular biology. The effect was first demonstrated in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, who showed gene transfer in Streptococcus pneumoniae. Avery, Macleod and McCarty call the uptake and incorporation of DNA by bacteria transformation. There are many variations on a common theme, but the key points are listed below. Check details with supplier of competent bacteria and note that variations in timings and volumes will vary with application and bacterial strain. There are four stages:Mix DNA/bacteria and incubate on ice- do not use an excessive amount of DNA, both in terms of concentration and actual volume (less than 1 µg and less than 10 µl). Note, protein (e.g. Ligase) will reduce transformation efficiency, but it is not always necessary to remove prior to transformation.Heat shock- necessary for DNA uptake. Time heat shock carefully- excessive heat shock will kill the bacteria and the transformation will fail.Recovery- prior to selecting for transformed bacteria with antibiotics, it is necessary to allow them to recover in rich medium (e.g. LB, SOC or 2YT) for 30-60 mins at 37 ûC.Selection- essential to isolate (as single colonies) the bacteria which have taken up DNA.This is usually performed on solid medium (LB-agar) in the presence of antibiotics. Cells are incubated at 37 ûC overnight. Competent bacteria are extremely fragile - always thaw slowly on ice and do not hold the base of the eppendorf tube. The compency of the bacteria is also important- sub-cloning efficiency means about 106 colonies are produced per µg of (purified) DNA. Library efficiency can mean in excess of 109 colonies produced per µg of (purified) DNA. For sub-cloning and mutagenesis is normally sufficient, although Library efficiency bacteria may be useful if problems arise.Materials and equipment:Competent cellsPre-chilled sterile eppendorfsDNA to be transformedWater bath at 42 °CAppropriate media and plates (see below)Protocol: 1Remove DH5α / BL21 / Topp10 (or other) cells from −80°C freezer and thaw on ice for 15-20 min.2. Aliquot 100 µl cells into pre-chilled sterile Eppendorfs. [NOTE: Subcloning efficiency DH5α are pre-aliquoted and kept in a box in the hallway shared –80°C freezer. If there are fewer than 15 tubes left in the box, please check to see if there are more cells available, otherwise make sure you order more.]3. Add 2-10 µl of ligation mix (or 1-5 µl of plasmid DNA) to each tube of 100 µl cells and incubate on ice for 20 minutes. (Up to 15 µl DNA can be used per transformation, but more DNA does not always translate to good transformation efficiency.)4. Heat shock at 42°C for 45 to 60 seconds.5. Add 900 µl LB or SOC medium and incubate for 1 hour at 37°C with shaking.6. Plate 200 µl on selective plates (either LB+Amp, LB+Kan etc.)Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satelite colonies will form. Plates/colonies can be stored for a few days at 4ûC if not to be used immediately.Blue white selectionMany vectors (such as pUC series) carry coding information for the first 146 amino acids of the b -galactosidase gene. Embedded in this coding region is the polycloning site (does not disrupt the reading frame of the gene) into which insert DNA is cloned. When expressed, this 146 amino acid fragment of b -galactosidase protein is incapable of acting on the chromogenic substrate (X-gal). But when expressed in appropriate host cells which expresses the carboxyl terminal fragment of the b -galactosidase protein, these two protein fragments can associate to form an enzymatically active protein. This is called as α-complementation and such cells turn blue when plated on plates containing X-gal. But if the insert DNA has gotten cloned in the polycloning site, it almost invariably results in production of an aminoterminal fragment that is not capable of a -complementation and hence those colonies remain white.•Spread the following on a LB (+appropriate antibiotic) plate (If you are going to plate 100ul of transformed cells): 100ul of 100mM IPTG + 40ul of X-gal (20mg/ml).•Perform transformation of ligation into the bacteria (note: make sure you are using the right host bacterial strain for the blue white selection, also see the background section below) and then spread them on the X-gal containing plates. •After overnight incubation, store the plate in 4 0 C for several hours. This allows the blue color to develop fully. Colonies that contain active b -galactosidase are pale blue in center and dense blue at their periphery. White colonies occassionally show a faint blue spot in the center, but these are colorless at the periphery.Stock solutions: •X-gal-X-gal is very expensive, and hence avoid wastage. X-gal stock solution can be prepared by dissolving 100mg of X-gal in 5ml of dimethyl formamide. X-gal is light sensitive and hence store in a glass or polypropylene containers wrapped with aluminium foil. Stock solution need to be store in -20 0 C •IPTG (Isopropyl Thio- b -D-Galactoside, F.W.= 238.31)-To get a 100mM stock solution, dissolve 0.238g in 8ml of dH2O and then bring the volume to 10ml with dH2O. Filter sterilize the solution by passing it through 0.22 micron filter, prepare 1ml aliquots and store them in -20 0 C. POLYMERASE CHAIN REACTION (PCR)The Polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.Materials & ReagentsA basic PCR set up requires several components and reagents. These components include:DNA template that contains the DNA region (target) to be amplified.Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand (2mM).Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.MgCl2 (25mM)Double Distilled Water. ProcedureTypically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon),agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.Application of PCRSelective DNA isolationAmplification and quantification of DNAIn diagnosis of diseasesRESULT AND DISCUSSIONDNA Isolation from BacteriaRESULT: Bacterial DNA was successfully isolated. The observed concentration by measuring the absorbance at 260 nm was 325µm. CTAB Method / Protocol for DNA Extraction from Plant Samples RESULT: Plant DNA was isolated successfully. Plasmid IsolationRESULT: Plasmid DNA was isolated successfully and dark bands were observed under UV-Transilluminator after Gel electrophoresis. RNA Isolation using Bio-Red RNA isolation kitRESULT: RNA was isolated successfully Protein Isolation From Plant SDS- PAGE OF PROTEINS Southern BlottingRESULT: DNA bands were observed under UV-Transilluminator.Competent Cell Preparation RESULT: Competent cell culture was obtained.TransformationRESULT: Blue and white colonies were observed in the competent cell culture showing transformed and non-transformed culture respectively. POLYMERASE CHAIN REACTION (PCR)