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Lab Manual Molecular Biology SHAHEED ZULFIKAR ALI BHUTTO INSTITUTE OF SCIENCE &TECHNOLOGY LAB MANUAL MOLECULAR BIOLOGY COURSE NO # ____...

Lab Manual Molecular Biology SHAHEED ZULFIKAR ALI BHUTTO INSTITUTE OF SCIENCE &TECHNOLOGY LAB MANUAL MOLECULAR BIOLOGY COURSE NO # ______ BS BIOSCIENCES/BS BIOTECHNOLOGY SHAHEED ZULFIKAR ALI BHUTTO INSTITUTE OF SCIENCE & TECHNOLOGY DEPARTMENT OF BIOSCIENCES 99, 100, 154 CLIFTON, KARACHI, PAKISTAN TEL: (021) 35824461-63 FAX: (021) 3583-0446 URL: WWW.SZABIST.EDU.PK Department of Biosciences, SZABIST Page 1 Lab Manual Molecular Biology CERTIFICATE This is to certify that Mr./ Ms.………………….S/o, D/o of ………………………………class ………………. Registration no ……………….has carried out the practical work as prescribed by the Institute for the year ………. Signature of Instructor................................. Department of Biosciences, SZABIST Page 2  Lab Manual Molecular Biology Codes of Conduct 1. Students must thoroughly read the assigned work. It will be assumed that they have studied the assigned work and have understood the majority of the material and technical terms before the start of the experiments. 2. All the original data have to be recorded in a Report sheet during the laboratory sessions. Data recording on rough sheets of papers are not allowed. In order to check for the originality of the data, ball pen or inerasable pen should be used. If correction has to be made, just cross it out. They must hand over the Report sheet to the demonstrator for their signature after data count. 3. For safety reasons, students are requested not to leave their equipment unattended during the lab. Session. In the case of special circumstances, please seek the support of the class teachers/demonstrators 4. All practical contribute to the final results of the sessional course. Thus, any absent laboratory session automatically means lost marks for the final grade. Under special circumstances (supported by documentary proof), e.g. illness and other reasonable causes a laboratory session may be re- scheduled upon approval of the head of the department. 5. During the class, students will be continuously assessed by performance test on each and every experiment. 6. To ensure your fellow students can proceed with their experiments in a degree of comfort and without undue noise and other disturbances, keep the noise level down and stay in your own laboratory bench area. Mobile phones should be switched off during the experiments Department of Biosciences, SZABIST Page 3 Lab Manual Molecular Biology RULES OF THE LABORATORY  Laboratory coats and covered shoes must be worn at all times in the laboratory.  No smoking, eating or drinking will be permitted in the laboratory.  Each student’s bench space must be wiped before and after the laboratory session with ethanol. Spills of stains or reagents must be wiped up immediately.  Accidents of any kind spilled strong acids/bases, cuts, burns, handling cultures e.g. breakages of tubes or spillages of cultures must be reported to the teacher immediately.  Properly label all materials to be used in the experiment.  All equipment must always be returned to their proper storage case when not in use. Any damage or defect in the microscope should be reported to your demonstrator promptly.  The equipment needed for the experiments will be found in your bench area. Be sure to inventory the equipment and take care in its use. No additional equipment will be provided.  Waste paper to be placed in bin provided in labs.  Never remove anything from the laboratory without your teacher's permission.  Never use your bare hands to transfer chemicals. Use a spatula instead.  Never put solids in the sink.  Wear safety glasses whenever necessary.  Always tie up your long hair.  Always wash hands after experiments.  Equipments’ log books should be properly maintained.  At the end of each lab session, please get your lab manuals signed by the instructor.  Final marks will be awarded after signing of lab manuals by HoD. Department of Biosciences, SZABIST Page 4 Lab Manual Molecular Biology LAB STANDARD OPERATIONS PROCEDURE (SOP) Bench area Clean bench area before and after use with disinfectant. Personal safety You must wear a lab coat. Wear coat only in the lab, transport separately outside of the lab (in a plastic bag). Wash hands with antibacterial soap before leaving the lab. No eating or drinking in the lab. Use aseptic technique for transfer of bacteria. This is to protect yourself as much as to ensure the purity of your culture. Protect hands with gloves and eyes with glasses when needed. The gloves provided in the lab are to be disposed of in dustbin. Long hair must be tied back. Closed toed shoes must be worn. Biohazards Know biosafety risk groups. Handle all cultures as potential pathogens. Never mouth pipette. Always use a pipette. If you spill a culture, cover the spill with paper towels. Gather up soaked towels and discard. Wipe area to dryness with fresh paper towels. Wash hands with soap and water. Place cultures on discard trolley. All cultures are autoclaved before disposing. Ethidium Bromide: Hazards and Precautions EB is a potent mutagen. It can be absorbed through the skin and is toxic upon acute exposure. EB is irritating to the eyes, skin, mucous membranes and upper respiratory tract. Pure EB should only be handled under a fume hood. Protective equipment including a lab coat, chemically resistant gloves and safety goggles (not glasses) should be worn. Nitrile gloves provide an effective barrier to short-term EB exposure. Lab workers should always wash their hands after removing gloves, even if they are sure the gloves were not punctured. EB should always be handled in the vicinity of an emergency eyewash and shower. UV-blocking eyewear should be worn whenever ultraviolet light is being used. Work should be done in a UV cabinet with shielding in place. Department of Biosciences, SZABIST Page 5 Lab Manual Molecular Biology Spill Response For small spills the following procedures should be followed: UV light can be used to locate the spilled material. If the EB is powder, use wet paper towels to wipe it up and dispose of the material properly. Follow the decontamination procedure below. If liquid is spilled, absorb the freestanding liquid with paper towels. Dispose of the material properly. Follow the decontamination procedure below. After cleaning the spill recheck the area with a UV light to be sure that all of the material has been removed Glassware (unbroken) Remove tape and pen markings (use alcohol) from glassware before placing on discard trolley. Used glassware should be rinsed and placed on the discard trolley. Rinsed test tubes should be placed in tray provided on the discard trolley. Used glass pipettes should be placed in pipette holders. Use extreme care with flammable solvents. Alcohol used to flame spread rod should never be positioned within 40 cm of flame. Never put a very hot spread rod into a beaker of alcohol. The alcohol may catch fire. Handle caustic (acids and bases) solutions with care. Never discard an acid or base greater than one molar down the sink. Discard in labelled glass containers provided. Use lots of water when discard caustic solutions (< 1M). These materials are disposed of through the university safety office. Never pour solvents down the sink (e.g. phenol, ether, chloroform, etc.). Discard in labelled containers provided. Sharps disposal Dispose of all sharps (needles etc.) in specified container. Dispose of syringe with needle attached - do not take apart. Do not replace the needle cap before disposing (high frequency of accidents occur when replacing cap). Sharp’s containers are autoclaved before disposing. OR in some labs, bacterial contaminated slides and Pasteur pipette disposal: Discard slides in designated plastic containers lined with clear plastic bags. Equipment operation Know how to operate equipment before use. DO NOT use equipment unless you know exactly how to operate the equipment. The demonstrator is always available to assist. Leave your bench area clean All equipment and supplies should be returned to original location. Department of Biosciences, SZABIST Page 6 Lab Manual Molecular Biology INDEX Examiner S.NO DATE EXPERIMENT PAGE Initial’s Genomic DNA Extraction by Manual 1. Method 2. Genomic DNA Extraction by Kit Method 3. Introduction to Spectrophotometer Nucleic Acid Analysis By 4. Spectrophotometry 5. Polymerase Chain Reaction 6. Agarose Gel Electrophoresis 7. Chromatography 8. Paper Chromatography 9. Thin Layer Chromatography 10. Calculations Department of Biosciences, SZABIST Page 7 Lab Manual Molecular Biology EXPERIMENT # 1 GENOMIC DNA EXTRACTION BY MANUAL METHOD INTRODUCTION: The fundamental steps of DNA purification are sample lysis and purification of the DNA from contaminants. There are myriad of protocols available for isolating DNA from organisms in the molecular lab. The more classical methods have remained essentially unchanged for decades, and the more modern methods involve kits that are commercially available. The best method for any particular application depends on these fundamental considerations: a. Where the DNA is isolated from will determine the cell lysis techniques used. b. The purity requirements of the intended use of the DNA being isolated will determine how many purification steps will be involved. The successful isolation of DNA requires methods that prevent nuclease degradation of the DNA. Some buffer constituents used to promote lysis and denaturation of nucleases includes: o Lysis Buffer There are different buffers available for different kind of tissues. CTAB lysis buffer is used mostly for plant cells. SDS lysis buffer is used when animal cells are being disrupted. But major components of the lysis or extraction buffer are same and performs same function in DNA Extraction. o Detergents CTAB buffer is a cationic buffer mostly used for plant cell disruption while SDS is anionic detergent used during animal cell lysis. Both SDS and CTAB interferes with membrane proteins and lipids bilayers leading to the disruption of the membranes of internal organelles, plasma membrane as well as nuclear membrane. o Proteinase K Sometimes added to cleave glycoproteins and to help the detergents to inactivate DNases. o Reducing agent β-mercaptoethanol acts as a reducing agents and cleaves the disulphide bridges present between different polypeptides of a proteins leading to the denaturation of the proteins. Department of Biosciences, SZABIST Page 8 Lab Manual Molecular Biology o Chelating agents EDTA acts as chelating agents and binds to Mg2+ ions which acts as co-factors for DNase. The unavailability of Mg2+ ions leads to the deactivation of DNase activity and hence saves the DNA from degradation. EDTA also weakens the membrane stability. o DNase DNase present in the cells can degrade the DNA. o Tris Buffer DNA is pH sensitive and can be degraded on pH change. Tris acts as pH stabilizer during cell lysis process. It maintains the pH at 8. o Salt – NaCl / KCl The cations Na+ or K+ binds to negative phosphate groups of DNA and makes it more stable in aqueous solution. In the absence of Na+ or K+, DNA molecules repel each other and do not allow grouping of DNA molecules.  DNA Precipitation Precipitation separates the DNA from broken down cell components during DNA extraction process. There are three major types of DNA separation from cell debris. o Ethanol precipitation Ice cold ethanol is added to the solution containing DNA and cell debris. Proteins gets dissolved in ethanol. Upon centrifugation, DNA is obtained in the form of pellet on the bottom of Eppendorf. Supernatant containing cell debris is discarded and DNA pellet is washed with ethanol to remove any salts or impurities. Centrifugation is done again, supernatant is discarded, pellet is air dried and then the pellet is suspended in pure water or suitable buffer for storage. Isopropanol can also be used instead of ethanol. Isopropanol is more effective in precipitation but it is less volatile than ethanol so more time is required for air drying. o RNases RNases are often added to a lysis buffer to remove contaminating RNAs, which can interfere with the intended use of the DNA being isolated. The number of steps in a cell lysis protocol should also be kept to a minimum, since any delays during this part of the DNA isolation procedure runs the risk of DNA degradation by nucleases in the cells. DNA will not be safely stabilized until it has been purified from all protein contaminants. In general, animal tissues are easily lysed, due to the fact that they have no cell wall, and a gently detergent treatment usually is sufficient to break open cells. Yeast Department of Biosciences, SZABIST Page 9 Lab Manual Molecular Biology and microbial cells, on the other hand, have rigid cell walls that must be weakened enzymatically before the cell will release its DNA. In the case of bacteria, lysozyme enzyme is added, while in the case of yeast a more complex mixture of enzymes must be used to degrade cell wall polymers. Plant cell walls are generally abraded mechanically by grinding frozen plant tissue, often with glass beads or sand and a mortar and pestle. The second phase of DNA isolation protocols is the purification of the DNA released from the cell from other components of the cell and the lysis buffer. The method you select for your application depends on the size and source of the DNA to be isolated. When plasmid DNA is being isolated from bacteria such as Escherichia coli (E. coli), an alkaline solution of SDS is sufficient to release plasmid DNA, leaving behind the genomic DNA still associated with the cellular debris. The genomic DNA is then conveniently removed from the plasmid DNA by a quick centrifugation step. Genomic DNA can frequently be rendered insoluble and quickly spooled from the lysed cells by addition of alcohol to the mixture. The spooled DNA can be transferred to a fresh buffer to re-dissolve the genomic DNA. For some applications, this low level of DNA purity will suffice. Often, though, there are proteins or polysaccharides (especially in plant sources of DNA) that co-precipitate with the DNA and interfere with subsequent enzymatic treatments. Classically, the further purification of DNA involves the removal of proteins by aqueous phenol solutions, followed by numerous alcoholic precipitation steps to remove traces of phenol from the isolated DNA. Alcohol precipitations of DNA also serve to concentrate the DNA into a smaller volume, and to purify the DNA from any water-soluble contaminants. The phenol extraction is an inefficient method of purification and suffers from a poor yield of DNA. Also, phenol reagents are unstable, and fresh solutions must be used or the quality of the reagent must be monitored, generally by observed changes in pH. This, along with safety concerns in the use of phenolic solutions, is a serious drawback in this method of DNA purification. An alternative procedure is the use of spin columns, which are small chromatography columns that purify the DNA from other solutes. While this procedure is more expensive than phenol extractions and alcohol precipitations, the purification and yields of product by spin columns are improved. In addition, the reagents used are more stable, so provide a more reliable, or robust method. The final DNA prepared with spin columns is free of protein and salt contaminants and can be used directly in restriction digests, southern blotting, and PCR applications. All components of this system are stable at room temperature for one year. Department of Biosciences, SZABIST Page 10 Lab Manual Molecular Biology Binding and elution from silica beads has become the method of choice for isolation of genomic DNA from animal tissues. A high concentration of chaotropes serves to bind nucleic acids to silica surfaces. The adsorption step to bind DNA to the silica particles is followed by wash steps, usually with salt/ethanol solutions, which will not interfere with the strong binding of nucleic acids but will wash away remaining impurities and excess chaotrope. Elution of DNA from silica columns requires the use of nuclease-free water or low ionic strength buffers such as TE. This is an advantage since it means that the isolated DNA can be used directly in further manipulations without further cleanup. A spin-column method that does not require the use of time-consuming and toxic phenol/chloroform extractions or ethanol precipitations. The final genomic DNA prep is free of protein and salt contaminants and can be used directly in restriction digests, Southern blotting, and PCR applications. A potential problem with the use of silica columns for the binding of DNA is the possibility of overloading the column with DNA, resulting in a wash-through of non-adsorbed DNA and reducing the overall yield of DNA. There is also some loss of material that does not elute from the silica resin. The smaller the DNA size is, the tighter is its interaction with silica surfaces. Although size is not a problem with isolations of genomic DNA, loading the silica resin with too little DNA can also lead to a low overall yield of DNA eluted from a silica column. PROTOCOL OF DNA EXTRACTION:  To the Eppendorf tube containing bacterial cells, 650μl of lysis buffer was added and the tubes were vortexed vigorously for 5 min to bring about the lysis of bacterial cells.  Tubes were centrifuged at 7800rpm for 10 min with subsequent transfer of 500μl of supernatant to the new Eppendorf containing 100μl of 3M Potassium-acetate buffer.  Tubes were inverted several times and centrifuged at 7800rpm for 3 min. Supernatant was transferred to a new tube containing 500μl of chilled isopropanol, tubes were inverted several times to make the bacterial DNA insoluble.  The contents of the Tubes were then centrifuged for 2 min at 7800rpm to obtain DNA pellet. Supernatant was discarded and 750ul of 70% ethanol was added to wash the DNA pellet and centrifuged at 7800rpm for 1 min.  Ethanol was removed from the tubes carefully and the DNA pellet was air-dried overnight in an incubator. Department of Biosciences, SZABIST Page 11 Lab Manual Molecular Biology  Once dried, 100 μl of sterile distilled water was added, vortexed a bit to dissolve the DNA and stored at -20°C for further experiments.  Lysis buffer was prepared by mixing together desired volumes of following buffers and reagents: A. Tris-HCl (0.1M, pH 8.0) B. Sodium EDTA (0.05M, pH 8.0) C. 1% SDS (w/v) D. RNAase A (10μg/ml) RESULTS AND DISCUSSION: Department of Biosciences, SZABIST Page 12 Lab Manual Molecular Biology EXPERIMENT # 2 GENOMIC DNA EXTRACTION BY KIT METHOD PROTOCOL: 1. Collect the sample of bacterial culture and centrifuge at 7800rpm for 5min. Remove supernatant. For gram-positive bacteria: Add 180 μl lysozyme solution (20 mg/ml lysozyme, 20 mM Tris-HCl, pH 8.0, 2.5 mM EDTA, 1% Triton X-100), suspend thoroughly and incubate the sample at 37°C for 30 minutes For gram-negative bacteria: Add 180ul of Universal Buffer Digestion to the sample. 2. Add 20ul of Proteinase K solution to sample and incubate at 56℃ for 1hour. 3. Add 200ul Universal Buffer BD, mix thoroughly by vortexing. Incubate at 70°C for 10 minutes. 4. Add 200µl ethanol (96-100%), mix thoroughly by vortexing. NOTE: If a gelatinous material appears at this step, vigorously shaking or vortexing is recommended. 5. Transfer the mixture from step 4 (including any precipitate) into the EZ-10 column (spin column) placed in a 2 ml collection tube. Centrifuge at 7800 rpm) for 1 minute. Discard the flow-through. Department of Biosciences, SZABIST Page 13 Lab Manual Molecular Biology 6. Add 500 µl Universal PW Solution, and centrifuge for 1 minute at 7800 rpm. Discard the flow-through. 7. Add 500 µl Universal Wash Solution, and centrifuge for 1 minute at 7800 rpm. Discard the flow-through. 8. Place the empty column in the micro-centrifuge and centrifuge for an additional 2 minutes at 7800 rpm to dry the EZ-10. Discard flow-through and transfer the spin column to a clean 1.5 ml centrifuge tube. NOTE: It is important to dry the membrane of the Ezup spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. 9. Add 100 µl Buffer CE directly onto the center part of EZ-10 membrane. Incubate at room temperature for 1 minute, and then centrifuge for 1 minute at 7800 rpm to elute the DNA. Basic Steps: DNA isolation and purification RESULTS AND DISCUSSION: Department of Biosciences, SZABIST Page 14 Lab Manual Molecular Biology EXPERIMENT # 3 INTRODUCTION TO SPECTROPHOTOMETER SPECTROPHOTOMETER:  A spectrophotometer is an instrument that measures the amount of light absorbed by a sample.  Spectrophotometer techniques are mostly used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer.  Scientist Arnold J. Beckman and his colleagues at the National Technologies Laboratory (NTL) invented the Beckman DU spectrophotometer in 1940. PRINCIPLE OF SPECTROPHOTOMETER: The spectrophotometer technique is to measure light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then on the display device. 1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into different wavelengths. 2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm. 3. The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength. Department of Biosciences, SZABIST Page 15 Lab Manual Molecular Biology INSTRUMENTATION OF SPECTROPHOTOMETER: The essential components of spectrophotometer instrumentation include: 1. A table and cheap radiant energy source  Materials that can be excited to high energy states by a high voltage electric discharge (or) by electrical heating serve as excellent radiant energy sources. 2. A monochromator, to break the polychromatic radiation into component wavelength (or) bands of wavelengths.  A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands. Prisms:  A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent  Two types of Prisms are usually employed in commercial instruments. Namely, 600 cornu quartz prism and 300 Littrow Prism. Grating:  Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions. 3. Transport vessels (cuvettes), to hold the sample  Samples to be studied in the ultraviolet (or) visible region are usually glasses (or) solutions and are put in cells known as “CUVETTES”.  Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz. 4. A Photosensitive detector and an associated readout system  Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it.  Radiation detectors generate electronic signals which are proportional to the transmitter light.  These signals need to be translated into a form that is easy to interpret.  This is accomplished by using amplifiers, Ammeters, Potentiometers and Potentiometric recorders. APPLICATIONS: Some of the major applications of spectrophotometers include the following: Department of Biosciences, SZABIST Page 16 Lab Manual Molecular Biology  Detection of concentration of substances  Detection of impurities  Structure elucidation of organic compounds  Monitoring dissolved oxygen content in freshwater and marine ecosystems  Characterization of proteins  Detection of functional groups  Respiratory gas analysis in hospitals  Molecular weight determination of compounds  The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations. Department of Biosciences, SZABIST Page 17 Lab Manual Molecular Biology EXPERIMENT # 4 NUCLEIC ACID ANALYSIS BY SPECTROPHOTOMETRY INTRODUCTION: The defacto method for quantitating nucleic acids that all other methods rely on is ultraviolet absorption. When other methods are used, a nucleic acid standard is prepared based on its absorbance at 260 nm, measured by a spectrophotometer. An advantage in the use of a spectrophotometer in nucleic acid quantitation lies in its high precision and the fact that sample is not destroyed by the assay and can be put to further analysis after quantitation. A word of caution, however, is needed: ultraviolet light damages DNA, causing mutations in the nucleotide sequences, so the duration of exposure to ultraviolet light should kept to a minimum. A spectroscopic analysis is very fast, another major reason for its routine use in a molecular lab for quantitating nucleic acids. A drawback to the use of a spectrophotometer lies in its low sensitivity and the large volumes that cuvettes come in (generally>0.5 mL). This means that small volumes of sample or low concentrations of nucleic acids are not easily assayed with a standard spectrophotometer. Another drawback to ultraviolet absorbance in nucleic acid quantitation lies in its interference by contaminating proteins. A spectrophotometer makes use of the transmission of light through a solution to determine the concentration of a solute within the solution. This is accomplished by placing a lamp on one side of a sample and a photocell or detector on the other side. All molecules absorb radiant energy at one wavelength or another, depending on the chemical types of functional groups they are comprised of. Those that absorb energy from within the visible spectrum are known as pigments. Proteins and nucleic acids absorb light in the ultraviolet range. Nucleic acid solutions are uncolored because the wavelengths that they absorb are outside the visible spectrum of light. With the aid of spectroscopy, the quantitative analysis of nucleic acids and proteins has established itself as a routine method in many laboratories. Both nucleic acids and proteins absorb in the ultraviolet range. Nucleic acids absorb strongly at 260 nm, proteins absorb more strongly at 280 nm. Converting OD into concentrations: The absorption of 1 OD (Optical Density or Absorbance Unit, a dimensionless quantity) is Department of Biosciences, SZABIST Page 18 Lab Manual Molecular Biology equivalent to approximately 50 µg/ml ds DNA, approximately 33 µg/ml ss DNA, 40 µg/ml RNA or approximately 30 µg/ml for oligonucleotides. Purity determination of DNA Interference by contaminants can be recognized by the calculation of the ratio. The ratio A260/A280 is used to estimate the purity of nucleic acid, since proteins absorb at 280 nm. Pure DNA should have a ratio of approximately 1.8, whereas pure RNA should give a value of approximately 2.0. Absorption at 230 nm reflects biological contaminants of the sample such as carbohydrates, peptides, salts, or proteins. Also, many chemicals commonly used in nucleic acid preparations, such as phenol, EDTA, and SDS, can be detected by their absorbance at 230 nm. Enzymes such as restriction endonucleases and polymerases are inhibited by low levels of phenol and SDS. DNA polymerases and DNase are notably sensitive to the presence of EDTA in nucleic acid samples. In the case of pure samples, the optical density of the sample (DNA) observed at ratio of 260/280 should lie in the range of 1.7-2.0). For calculating the concentration of DNA, the below mentioned formula is used: DNA Concentration = Abs 260 x Dilution factor x 50 (µg/ml or ng/µl) Glass absorbs UV light and thus is inappropriate for use in a UV spectrophotometer cuvette. Quartz and UV-transparent plastic cuvettes and Quartz Cuvettes are available for measuring absorbance in the UV range. RESULTS AND DISCUSSION: Department of Biosciences, SZABIST Page 19 Lab Manual Molecular Biology EXPERIMENT # 5 POLYMERASE CHAIN REACTION (PCR) PCR is probably the most widely used molecular biology technique. Its applications includes: diagnostics, tracing evolutionary relationships, cloning of genomic DNA and cloning of expressed DNA sequences. Because there are so many different uses for PCR, and because each unique primer pair can have varying optimal reaction conditions, it is impossible to just define one PCR protocol. Department of Biosciences, SZABIST Page 20 Lab Manual Molecular Biology PCR REACTION COMPONENTS: H20: PCR reactions can be negatively affected by the use of water with any organic compounds present. Use only water that is purchased from a chemical supply company that does a full analysis of every batch of water. Remember to use this water not only to set up reactions, but also to prepare and dilute any stock solutions. Buffer mix: The buffer is usually supplied with the polymerase being used. It provides the optimal salt concentration, pH and cofactors required by the polymerase. Generally, it is supplied in a 10X concentration and diluted 1/10 in the PCR reaction. dNTP: dNTPs are required in order for the polymerase to synthesize DNA. Generally, we dilute these from concentrated stocks (normally to 10 mM each of A, C, G and T). The final concentration of dNTP in the reaction is variable from procedure to procedure but generally is around 0.2 mM MgCl 2 or other co-factor: MgCl2 is required as a co-factor for most DNA polymerases used in PCR reactions. A stock solution (usually 50 mM) is usually supplied by the polymerase manufacturer. If you need to make your own stock, make sure to use molecular grade water and molecular grade MgCl2. Optimal reaction concentration is generally between 0.5 to 2 mM and frequently is determined empirically. The MgCl2 concentration influences the specificity of primer/template binding; thus altering the concentration can improve PCR specificity. Some polymerases require different co- factors. DNA primers: Most protocols use a primer pair. These are made using a DNA synthesizer (synthesis is outsourced). Primers are supplied desalted and dry and are resuspended at a concentration at least 10 fold higher than the working concentration with molecular grade water. Generally working concentration is 25 µM and the final concentration in the PCR reaction is 1 µM. Note that primers are unstable at less than 100 µm, so only make up small volumes of the working concentration and use diluted primers within a week. Department of Biosciences, SZABIST Page 21  Lab Manual Molecular Biology Polymerase: There are too many different polymerases to even try. Most polymerases used in PCR are either cloned versions of the original Taq DNA polymerase or are modified versions of Taq DNA polymerase. One common modification of Taq is to render the enzyme inactive until it reaches a high temperature. This “hot-start Taq” is frequently used to prevent miss-priming, particularly when working with entire genome. Template DNA: Optimal amount of template varies depending on source and how clean it is and the proportion of the total DNA that is targeted. This usually has to be determined empirically but a good starting point is 20 - 50 ng for cDNA, 0.1 – 1 ng for plasmid DNA and 50 to 100 ng for genomic DNA. Too much or too little DNA can result in undesirable result. Other components: Depending on the application, there are numerous additional components that might be needed. Refer to the manufacture’s instructions for the specific polymerase enzyme and the application. Note PCR is a very sensitive technique, thus, you must take care to prevent any contamination of PCR reactions with unwanted DNA, in particular, previously produced PCR products. PCR products can become aerosolized when tubes are opened for gel loading, and then settle on lab surfaces. They also can easily get into pipettes. DNA fragments are relatively stable so touching a contaminated surface and then touching clean tips/tubes can transfer unwanted DNA into you PCR reaction, as can using contaminated pipettes. DNA is not fully destroyed by autoclaving. It is destroyed by UV treatment, or treatment with basic solutions. Department of Biosciences, SZABIST Page 22 Lab Manual Molecular Biology BASIC PCR REACTION: Stock Working Volume/Reaction Volume/ 5 Reagents (Concentration) (Concentration) (50 µl) Reactions PCR Buffer 10 X 1X 5 µl 25 µl MgCl2 50 mM 1.5 mM 1.5 µl 7.5 µl dNTPs 25 mM 0.2 mM 0.4 µl 2.0 µl Primer ‘F’ 20 µM 0.6 µM 1.5 µl 7.5 µl Primer ‘R’ 20 µM 0.6 µM 1.5 µl 7.5 µl Taq Polymerase 5 Units/µl 2.5 Units 1.2 µl 6 µl DNA 50ng/µl 250 ng 12.5 µl 62.5 µl H2O - - 26.4 µl 132 µl Total 50 µl 250 µl 1. Using the manufacturer’s instructions or a published protocol or the above guidelines, work out how to set up a single reaction and a master mix if needed. It is a good idea to program the thermocycler at this time as well. (make a pool of all the components except the DNA, make the pool 10% larger then need i.e. for 10 reactions make a pool of all components times 11 – this allows for pipette precision and accuracy errors). 2. Thaw all components and keep them cold in a cold block or ice bath. Set up the master mix (add the water first and the enzyme last). 3. Aliquot the master mix to individual tubes. Use only pipettes designated for PCR reaction set-up. 4. Add the DNA template; no mixing is necessary. 5. Place tubes in a thermocycler and start your program running. Cycling conditions are application and primer dependent; here is a relatively standard program. 35 cycles of 94 ° C for 1 min Tm of primers +/- 2 ° C for 1 min 72 ° C for 1 min 1 cycle of 72 ° C for 5 min (final extension) Hold till 6 °C until products can be removed form machine 6. When cycling is complete, run an aliquot (between 2 and 10 µl) of each reaction on an Department of Biosciences, SZABIST Page 23 Lab Manual Molecular Biology agarose gel of appropriate concentration. NOTE: The gel running and PCR set up areas are separated to keep the PCR area free of DNA contamination. Never open your tubes containing PCR product in the lab housing the PCR set up area. RESULTS AND DISCUSSION: Department of Biosciences, SZABIST Page 24 Lab Manual Molecular Biology EXPERIMENT # 6 AGAROSE GEL ELECTROPHORESIS Gel electrophoresis refers to using a gel as sieving medium during electrophoresis. Gel electrophoresis is most commonly used for separation of biological macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein; however, gel electrophoresis can be used for separation of nanoparticles. Electrophoresis refers to the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field. They act as a sieving medium, which separate the molecules on the basis of their size by hindering their passage through pore size. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR. It is also a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization. MIGRATION OF DNA FRAGMENTS IN AGAROSE: Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs. Migrating of pattern of different forms of molecules differ because of their size, shape, mobility shift. Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image below shows an ethidium- stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane. Department of Biosciences, SZABIST Page 25 Lab Manual Molecular Biology THE RATE OF MIGRATION OF DNA THROUGH AGAROSE GELS: The following factors determine the rate of migration of DNA through agarose gels:  The molecular size of the DNA. Molecules of double – stranded DNA migrate through gel matrices at rates that inversely proportional to the log10 of the number of base pairs (Helling et al. 1974). Larger molecules migrate more slowly because of greater frictional drag and because they worm their way through the pores of the gel less efficiently than smaller molecules.  The concentration of agarose. By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose Department of Biosciences, SZABIST Page 26 Lab Manual Molecular Biology facilitate separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. The above image shows migration of a set of DNA fragments in three concentrations of agarose. All of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.  The conformation of the DNA. Superhelical circular (form I), nicked circular (form II), and linear (form III) DNAs migrate through agarose gels at different rates (Throne 1966, 1967). The relative mobilities of the three forms depend primarily on the concentration and type of agarose used to the three forms depend primarily on the concentration and type of agarose used to make the gel, but they are also influenced by the strength of the applied current, the ionic strength of the buffer, and the density of super helical twists in the form I DNA (Johnson and Grossman 1977). Under some conditions, DNA migrates faster than form III DNA; under other conditions, the order is reversed. In most cases, the best way to distinguish between the different conformational forms of DNA is simply to include in the gel a sample of untreated circular DNA and a sample of the same DNA that has been linearized by digestion with a restriction enzyme that cleaves the DNA in only one place.  The presence of ethidium bromide in the gel and electrophoreses buffer. Intercalation of ethidium bromide causes a decrease in the negative charge of the double stranded DNA and an increase in both its stiffness and length. The rate of migrations of the linear Department of Biosciences, SZABIST Page 27 Lab Manual Molecular Biology DNA-dye complex through gels is consequently retarded by a factor of approx – 15% (Sharp et al. 1973).  The applied voltage. At low voltages, the rate of migration of linear DNA fragments is proportional to the voltage applied. However, as the strength of the electric field is raised, the mobility of high-molecular-weight fragments increase differentially. Thus, the effective range of separation in agarose gels decreases as the voltage is increased. To obtain maximum resolution of DNA fragments >2 kb in size, agarose gels should be run at no more than 5-8 V/cm.  The electrophoresis buffer: The electrophoresis mobility of DNA is affected by the composition and ionic strength of the electrophoresis buffer. In the absence of ions (e.g., if water is substituted for electrophoresis buffer in the gel or in the reservoirs), electrical conductivity is minimal. ELECTROPHORESIS BUFFERS: Several different buffers are available for electrophoreses of native, double-stranded DNA. These contain Tris-acetate and EDTA (pH 8.0; TAE), Tris-borate (TBE), or Tris-phosphate (TPE) at a concentration of approximately 50 mM (pH 7.5-7.8). Electrophoresis buffers are usually made up as concentrated solutions, and stored at room temperature. All of these buffers work well, and the choice among them is largely a matter of personal preference. TAE has the lowest buffering capacity of the three and will become exhausted if electrophoresis is carried out for prolonged periods of time. When this happens, the anodic portion of the gel becomes acidic and the bromophenol blue migrating through the gel toward the anode changes in color from bluish-purple to yellow. This change begins at pH 4.6 and in complete at pH 3.0. Exhaustion of TAE can be avoided by periodic replacement of the buffer during electrophoresis or by recirculation of the buffer between the two reservoirs. Both TBE and TPE are slightly more expensive than TAE, but they have significantly higher buffering capacity. Double stranded linear DNA fragments migrate~ 10 % faster through TAE than through TBE or TPE; the resolving power of TAE is slightly better than TBE or TPE for high- molecular-weight DNAs and worse for low molecular-weight DNAs. This difference probably explains the observation that electrophoresis in TAE yields better resolution of DNA fragments Department of Biosciences, SZABIST Page 28 Lab Manual Molecular Biology in highly complex mixtures such as mammalian DNA. For this reason, Southern blots used to analyze complex genomes are generally derived from gels prepared in and run with TAE as the electrophoresis buffer. The resolution of supercoiled DNAs is better in TAE than in TBE. Table 1: Electrophoresis Buffers BUFFER WORKING SOLUTION STOCK SOLUTION/LITER 50x 1x 242 g of Tris base TAE 40 mMTris-acetate 57.1 ml of glacial acetic acid 1mM EDTA 100 ml of 0.5 M EDTA (pH 8.0) 1x 10x 90 mMTris-phosphate 108 g of Tris base TPE 15.5 ml of phosphate acid (85%, 1.679 g/ml) 2 mM EDTA 40 ml of 0.5 M EDTA (pH 8.0) 5x 0.5x 54 g of Tris base a TBE 45 mMTris-borate 27.5 g of boric acid 1 mM EDTA 20 ml of 0.5 M EDTA (pH 8.0) TBE is usually made and stored as a 5x or 10x stock solution. The pH of the concentrated stock buffer should be 8.3. Dilute the concentrated stock buffer just before use and make the gel solution from the same concentrated stock solution. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more Department of Biosciences, SZABIST Page 29 Lab Manual Molecular Biology stable because the solutes do not precipitate during storage. Passing the concentrated buffer stocks through a 0.45-µm filter can prevent or delay formation of precipitates. Table 2: 6X Gel Loading Buffer STORAGE 6X BUFFER TEMPERATURE 0.25% bromophenol blue 0.25% xylene cyanol FF 4OC 30% glycerol in H2O GEL-LOADING BUFFERS: Gel-loading buffers are mixed with the samples before loading into the slots of the gel. These buffers serve three purposes: They increase the density of the sample with the help of glycerol, ensuring that the DNA sinks evenly into the well; they add color to the sample, thereby simplifying the loading process; and the contain dyes that, in an electric field, move toward the anode at predictable rates. Bromophenol blue migrates through agarose gels approximately 2.2- fold faster than xylene cyanol FF, independent of the agarose concentration. Bromophenol blue migrates through agarose gels run in as 0.5X TBE at approximately the same rate as linear double stranded DNA 300 bp in length, whereas xylene cyanol FF migrates at approximately the same rate as linear double stranded DNA 4 kb in length. These relationships are not significantly affected by the concentration of agarose in the gel over the range of 0.5-1.4%. The type of loading dye to use is a matter of personal preference. MATERIALS:  Buffers and Solutions Agarose gels are cast by melting the agarose in the presence of the desired buffer until a clear, transparent solution is achieved. The melted solutions is then poured into a mold and allowed to Department of Biosciences, SZABIST Page 30 Lab Manual Molecular Biology harden. Upon hardening, the agarose forms a matrix, the density of which is determined by the concentration of the agarose. Electrophoresis buffer (usually 1x TBE). Ethidium bromide 6x Gel-loading dye  Nucleic Acids and Oligonucleotides DNA samples DNA size standards Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or bacteriophage DNA of known sequence. Alternatively, they are produced by ligating a monomer DNA fragment of known size into a ladder of polymeric forms size standards for both agarose and polyacrylamide gel electrophoreses may be purchased from commercial sources or they can be prepared easily in the laboratory. It is a good idea to have two size ranges of standards including a high molecular weight range from 1 kb to > 20 kb and a low molecular weight range from 100 bp to 1000 bp. A stock solution of size standards can be prepared by dilution with a gel-loading buffer and then used as needed in individual electrophoresis experiments.  Equipment for agarose gel electrophoresis Clean, dry horizontal electrophoresis apparatus with chamber and comb or clean dry glass plates with appropriate comb.  Gel sealing tape Common types of lab tape, such as time tape or VWR lab tape, are appropriate for sealing the ends of the agarose gel during pouring.  Microwave oven or Boiling water bath Department of Biosciences, SZABIST Page 31 Lab Manual Molecular Biology PREPARATION OF SAMPLES: Mix 3 µl of genomic DNA and 5 µl of loading dye. PREPARATION OF 1% AGAROSE GEL (100 ml): 1. Weigh 1gm of agarose using electronic balance and transfer the agarose in a reagent bottle. 2. Add 100 ml of 1X TBE Buffer. (100 ml 10X and 900 ml water) 3. Heat till the homogenous solution form. 4. Add 5 µl of Ethidium Bromide. 5. Pour the gel into gel casting tray. Seal the edges of a clean, dry glass plate (or the open ends of the plastic tray supplied with electrophoresis apparatus) with tape to form a mold. Set the mold on a horizontal section of the bench. 6. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5 – 1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold. 7. Allow the gel to set completely (30-15 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape. Mount the gel in the electrophoresis tank. 8. After the gel solidifies, put it in gel tank. 9. Load the samples. Cover the lid of electrophoresis assembly. 10. Turn on power pack using 100 Volts, for 1 hour. 11. Observe the results using gel documentation system. RESULTS AND DISCUSSION: Department of Biosciences, SZABIST Page 32 Lab Manual Molecular Biology Figure: Pouring of Horizontal agarose gel Department of Biosciences, SZABIST Page 33 Lab Manual Molecular Biology EXPERIMENT # 7 CHROMATOGRAPHY CHROMATOGRAPHY:  Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.  The Russian botanist Mikhail Tswett coined the term chromatography in 1906.  The first analytical use of chromatography was described by James and Martin in 1952, for the use of gas chromatography for the analysis of fatty acid mixtures.  A wide range of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties to separate materials.  It is a powerful separation tool that is used in all branches of science and is often the only means of separating components from complex mixtures. PRINCIPLE OF CHROMATOGRAPHY: Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase.  The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.  Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster. Three components thus form the basis of the chromatography technique. 1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”. 2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.” 3. Separated molecules Department of Biosciences, SZABIST Page 34 Lab Manual Molecular Biology The type of interaction between the stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on the separation of molecules from each other. TYPES OF CHROMATOGRAPHY: Substances can be separated on the basis of a variety of methods and the presence of characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.  This leads to different types of chromatography techniques, each with their own instrumentation and working principle.  For instance, four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion.  Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. CHROMATOGRAPHY TECHNIQUES: 1. Column chromatography 2. Ion-exchange chromatography 3. Gel-permeation (molecular sieve) chromatography 4. Affinity chromatography 5. Paper chromatography 6. Thin-layer chromatography 7. Gas chromatography (GS) 8. Dye-ligand chromatography 9. Hydrophobic interaction chromatography 10. Pseudo affinity chromatography 11. High-pressure liquid chromatography (HPLC) APPLICATIONS OF CHROMATOGRAPHY: Pharmaceutical sector  To identify and analyze samples for the presence of trace elements or chemicals.  Separation of compounds based on their molecular weight and element composition. Department of Biosciences, SZABIST Page 35 Lab Manual Molecular Biology  Detects the unknown compounds and purity of mixture.  In drug development. Chemical industry  In testing water samples and also checks air quality.  HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils.  In various life sciences applications Food Industry  In food spoilage and additive detection  Determining the nutritional quality of food Forensic Science  In forensic pathology and crime scene testing like analyzing blood and hair samples of crime place. Molecular Biology Studies  Various hyphenated techniques in chromatography such as EC-LC-MS are applied in the study of metabolomics and proteomics along with nucleic acid research. HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation, and Enzyme Purification and also in various departments like Fuel Industry, biotechnology, and biochemical processes. Department of Biosciences, SZABIST Page 36 Lab Manual Molecular Biology EXPERIMENT # 8 PAPER CHROMATOGRAPHY Paper chromatography (PC) is a type of a planar chromatography whereby chromatography procedures are run on a specialized paper. PC is considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds. It was first introduced by German scientist Christian Friedrich Schonbein (1865). TYPES OF PAPER CHROMATOGRAPHY: 1. Paper Adsorption Chromatography Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase. 2. Paper Partition Chromatography Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent In general paper chromatography mostly refers to paper partition chromatography. PRINCIPLE OF PAPER CHROMATOGRAPHY: The principle of separation is mainly partition rather than adsorption. Substances are distributed between a stationary phase and mobile phase. Cellulose layers in filter paper contain moisture Department of Biosciences, SZABIST Page 37 Lab Manual Molecular Biology which acts as stationary phase. Organic solvents/buffers are used as mobile phase. The developing solution travels up the stationary phase carrying the sample with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase. INSTRUMENTATION OF PAPER CHROMATOGRAPHY: 1. Stationary phase & papers used 2. Mobile phase 3. Developing Chamber 4. Detecting or Visualizing agents Department of Biosciences, SZABIST Page 38 Lab Manual Molecular Biology 1. STATIONARY PHASE AND PAPERS  Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc  In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose. Other modified papers  Acid or base washed filter paper  Glass fiber type paper.  Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.  Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography.  Impregnation of silica, alumna, or ion exchange resins can also be made. 2. PAPER CHROMATOGRAPHY MOBILE PHASE  Pure solvents, buffer solutions or mixture of solvents can be used. Examples- Hydrophilic mobile phase  Isopropanol: ammonia:water 9:1:2  Methanol : water 4:1  N-butanol : glacial acetic acid : water 4:1:5 Hydrophobic mobile phases  dimethyl ether: cyclohexane kerosene : 70% isopropanol  The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated.  If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied. 3. CHROMATOGRAPHIC CHAMBER  The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most.  They are available in various dimensional size depending upon paper length and development type. Department of Biosciences, SZABIST Page 39 Lab Manual Molecular Biology  The chamber atmosphere should be saturated with solvent vapor. PROTOCOL: In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include: 1. SELECTION OF SOLID SUPPORT Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of sample and favouring good rate of movement of solvent. 2. SELECTION OF MOBILE PHASE Different combinations of organic and inorganic solvents may be used depending on the analyte. Example. Butanol: Acetic acid: Water (12:3:5) is suitable solvent for separating amino-acids. 3. SATURATION OF TANK The inner wall of the tank is wrapped with the filter paper before solvent is placed in the tank to achieve better resolution. 4. SAMPLE PREPARATION AND LOADING If solid sample is used, it is dissolved in a suitable solvent. Sample (2-20ul) is added on the base line as a spot using a micropipette and air dried to prevent the diffusion. 5. DEVELOPMENT OF THE CHROMATOGRAM Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited until the solvent front reaches near the edge of the paper. Different types of development techniques can be used: ASCENDING DEVELOPMENT  Like conventional type, the solvent flows against gravity.  The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. DESCENDING TYPE  This is carried out in a special chamber where the solvent holder is at the top.  The spot is kept at the top and the solvent flows down the paper. Department of Biosciences, SZABIST Page 40 Lab Manual Molecular Biology  In this method solvent moves from top to bottom so it is called descending chromatography. ASCENDING – DESCENDING DEVELOPMENT  A hybrid of above two techniques is called ascending-descending chromatography.  Only length of separation increased, first ascending takes place followed by descending. CIRCULAR / RADIAL DEVELOPMENT  Spot is kept at the centre of a circular paper.  The solvent flows through a wick at the centre & spreads in all directions uniformly. DRYING OF CHROMATOGRAM After the development, the solvent front is marked and the left to dry in a dry cabinet or oven. DETECTION Colourless analytes detected by staining with reagents such as iodine vapour, ninhydrin etc. Radiolabeled and fluorescently labeled analytes detected by measuring radioactivity and florescence respectively. Rf VALUES: Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as other parameters such as the type of paper and the exact composition of the solvent are constant. The distance travelled relative to the solvent is called the Rf value. Thus, in order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, “Rf value” is calculated for each separated component in the developed chromatogram. An Rf value is a number that is defined as distance traveled by the component from application point. Department of Biosciences, SZABIST Page 41 Lab Manual Molecular Biology APPLICATIONS OF PAPER CHROMATOGRAPHY:  o check the control of purity of pharmaceuticals,  For detection of adulterants,  Detect the contaminants in foods and drinks,  In the study of ripening and fermentation,  For the detection of drugs and dopes in animals & humans  In analysis of cosmetics  Analysis of the reaction mixtures in biochemical labs. ADVANTAGES OF PAPER CHROMATOGRAPHY:  Simple  Rapid  Paper Chromatography requires very less quantitative material.  Paper Chromatography is cheaper compared to other chromatography methods.  Both unknown inorganic as well as organic compounds can be identified by paper chromatography method.  Paper chromatography does not occupy much space compared to other analytical methods or equipment.  Excellent resolving power Department of Biosciences, SZABIST Page 42 Lab Manual Molecular Biology LIMITATIONS OF PAPER CHROMATOGRAPHY:  Large quantity of sample cannot be applied on paper chromatography.  In quantitative analysis paper chromatography is not effective.  Complex mixture cannot be separated by paper chromatography.  Less Accurate compared to HPLC or HPTL OBSERVATIONS: RESULT: Department of Biosciences, SZABIST Page 43 Lab Manual Molecular Biology EXPERIMENT # 9 THIN LAYER CHROMATOGRAPHY  Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.  In this physical method of separation, the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Depending upon the stationary phase and mobile phase chosen, they can be of different types.  Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase. PRINCIPLE OF THIN LAYER CHROMATOGRAPHY (TLC):  Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. Department of Biosciences, SZABIST Page 44 Lab Manual Molecular Biology  After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.  It is thus based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed. The components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster.  Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character is identified by means of suitable detection techniques. COMPONENTS OF THIN LAYER CHROMATOGRAPHY (TLC): TLC system components consists of: 1. TLC plates, preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size. 2. TLC chamber- This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free. 3. The stationary phase is applied onto the plate uniformly, and then allowed to dry and stabilize. These days, however, ready-made plates are more commonly used 4. Mobile phase- This comprises of a solvent or solvent mixture. The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase. 5. A filter paper- This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase. Department of Biosciences, SZABIST Page 45 Lab Manual Molecular Biology PROTOCOL: 1. With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots. 2. Then, samples solutions are applied on the spots marked on the line in equal distances. 3. The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom. 4. A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect). 5. Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid. 6. The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent) for development. 7. Sufficient time is given for the development of spots. 8. The plates are then removed and allowed to dry. 9. The sample spots are then seen in a suitable UV light chamber, or any other methods as recommended for the given sample. Department of Biosciences, SZABIST Page 46 Lab Manual Molecular Biology 10. The Rf value is then calculated for the spots. Some common techniques for visualizing the results of a TLC plate include 1. UV light 2. Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine 3. KMnO4 stain (organic molecules) 4. Ninhydrin Reagent: often used to detect amino acids and proteins APPLICATIONS OF THIN LAYER CHROMATOGRAPHY (TLC): 1. In monitoring the progress of reactions 2. Identify compounds present in a given mixture 3. Determine the purity of a substance.  Analyzing ceramides and fatty acids  Detection of pesticides or insecticides in food and water  Analyzing the dye composition of fibers in forensics  Assaying the radiochemical purity of radiopharmaceuticals  Identification of medicinal plants and their constituents ADVANTAGES OF THIN LAYER CHROMATOGRAPHY (TLC): Department of Biosciences, SZABIST Page 47 Lab Manual Molecular Biology  It is a simple process with a short development time.  It helps with the visualization of separated compound spots easily.  It helps in isolating of most of the compounds.  The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation).  The purity standards of the given sample can be assessed easily.  It is a cheaper chromatographic technique. LIMITATIONS OF THIN LAYER CHROMATOGRAPHY (TLC):  It cannot tell the difference between enantiomers and some isomers.  In order to identify specific compounds, the Rf values for the compounds of interest must be known beforehand.  TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques. OBSERVATIONS: RESULT: Department of Biosciences, SZABIST Page 48 Lab Manual Molecular Biology CALCULATIONS The concentration of a solution may be defined as the amount of solute present in the given quantity of the solution. Most commonly, the concentration of solutions are expressed in four ways. 1. Concentration in moles per litre, molar concentration or molarity (mol/L or mol L-1 or M) 2. Concentration by percentage (either % w/v or % v/v or sometimes %w/w) 3. Concentration in grams per litre (g/L or g L-1) 4. Preparing solutions by dilution 5. Preparing saturated solutions MOLAR SOLUTIONS: Molarity is number of moles of solute dissolved in 1 liter of the solution. A molar solution denoted by “M”. A molar solution (1 M) contains 1gram mole of the solute dissolved in water/solvent and volume made up to 1 liter. A millimolar (mM) solution contains 1/1000 of a mole of solute dissolved in the solvent and volume made up to 1 liter. Example: Prepare 1M sodium hydroxide solution. The molecular weight of sodium hydroxide is 40 grams. To calculate the amount required to make the above solution, use the following formula: Amount (in grams) = Molarity x Molecular Weight (gm) x volume (ml) 1000 Put the values of Molarity as 1, molecular weight as 40, volume as 1000, in the above formula in order to calculate the required amount. The above formula can also be used to calculate the resultant molarity if the amount of solute is given. Converting Percent Solution to Molarity: For conversion of percent solution to molarity or vice versa, use the following formula: Department of Biosciences, SZABIST Page 49 Lab Manual Molecular Biology Molarity = Percent Solution x 10 Molecular Weight PERCENT (%) SOLUTIONS: Percent (%) solutions refer to the amount of solute (in grams) in 100 ml of solvent. Example: 10% aqueous sodium chloride solution. To make this solution, weigh accurately 10 grams of sodium chloride and dissolve in 60-70 ml of water and then make up the volume up to 100 ml. There are four ways of expressing percentage composition of a solution. a. Weight by weight (w/w) percent solution: A solution in which solute (in grams) dissolved per 100 grams of solution (not the solvent). A 20% w/w solution contains 20grams of solute in 80 grams of solvent. These types of percent solutions are usually used in extraction and purification protocols. b. Weight by volume (w/v) percent solution: A solution in which solute (in grams) dissolved per 100 ml of solution (not the solvent). A 20% w/v solution contains 20gram of solute dissolved in a final volume of 100 ml solution. For example, 2%w/v solution of sodium chloride would be prepared from 2 g of sodium chloride dissolved in water and made up to a volume of 100 mL. c. Volume by volume (v/v) percent solution: A solution in which solute (in ml) dissolved per 100 ml of solution (not the solvent). A 20% v/v solution contains 20 ml of solute dissolved in a final volume of 100 ml solution. For example, 5%v/v aqueous solution of ethanol would be prepared by taking 5 mL of pure ethanol and diluting this with 95mL water to a volume of 100 mL. PRACTICE QUESTIONS: 1. What is the molarity of a solution that contains 5.5 moles of sodium chloride in 10.5 liters of solution? Department of Biosciences, SZABIST Page 50 Lab Manual Molecular Biology 2. If I have 3.50 grams of sodium chloride in 1250 mL of a solution, what is the concentration? 3. Explain how you would make 450 mL of a 0.25 M calcium chloride solution. 4. You want to make 60 ml of a 20% NaCl solution. How much NaCl do you need and how do you make this solution? Department of Biosciences, SZABIST Page 51 Lab Manual Molecular Biology 5. If you put 40 g of sugar into a final volume of 1.0 L of dH2O (distilled), what percent sugar is your solution? 6. How much sodium hypochlorite(bleach) is needed to make 230 ml of 30% bleach solution? 7. What is the molarity of a solution made when you dilute 35 grams of sodium carbonate to a volume of 3400 mL? Department of Biosciences, SZABIST Page 52 Lab Manual Molecular Biology 8. 100 g of active ingredient powder is mixed with 500 mL normal saline. What is the final concentration [w/v]? 9. 180 mL of active ingredient is added to 820 mL of an alcohol-based solution. What is the final strength [v/v]? 10. For each of the following, indicate the mass (g) of reagent needed to prepare 125 ml of the indicated percent solution: 10% NaCl 4.5% Na2PO4 Department of Biosciences, SZABIST Page 53 Lab Manual Molecular Biology 2.5% Citric acid 11. What mass of solute is needed to prepare 500 mL of 0.350 M C6H12O6? 12. What is the Molarity of a 2% NH2SO4 solution? 13. Convert 0.03μg into nanograms. 14. Convert 0.0025ml into μl 15. Calculate how much NaCl is used to make 300ml of 450mM solution. Department of Biosciences, SZABIST Page 54