BIO206 Lab 1 Manual.pdf

BIO206 Laboratory 1 Spectrophotometric analysis of DNA and proteins Before Lab: Carefully review the safety guidelines from the Week 1 Introductory session (Lab 0). You will not be permitted to participate in labs without appropriate safety clothing. Read and understand all lab exer...

BIO206 Laboratory 1 Spectrophotometric analysis of DNA and proteins Before Lab: Carefully review the safety guidelines from the Week 1 Introductory session (Lab 0). You will not be permitted to participate in labs without appropriate safety clothing. Read and understand all lab exercises and their background theory. There are Pre-lab Quizzes for all Labs including this one. After Lab: Hand in Laboratory 1 Worksheet to Quercus before 10 pm on the day after your lab Lab 1 outline Accurately quantifying concentrations of DNA and proteins in solution is a basic exercise in molecular biology labs. During these experiments, you must handle small volumes of reagents using micropipettes to do dilutions and serial dilutions. Lab 1 is a three-part lab. Part A will train you how to use micropipettes. In Part B, you will use measure the concentration of an unknown DNA solution using Salmon sperm DNA as an example. Part C is a read-only lab which reviews spectrophotometry of protein solutions. Part A: Introduction to Micropipettes Part B: Quantification of DNA solution using spectrophotometry Part C: Quantification of protein solution using spectrophotometry (Read-only, still testable material in Quizzes etc.) Safety guidelines for Lab 1 Salmon sperm DNA is typically harmless to human body, but sill handle them with caution. Rinse with running water immediately and thoroughly if it gets on your skin. BIO206 Fall 2024 / University of Toronto Mississauga 1 Part A1: Introduction to Micropipettes / Practice exercise In a molecular biology lab, you routinely handle volumes of reagents in the microliter (μL) range. One microliter is 1/1000 of 1 milliliter (mL, 1 mL = 1000 μL), a volume too small to be measured using a graduated cylinder. Micropipettes are used to handle these small volumes with high accuracy (see Appendix A for metric conversions). Micropipettes comes in different sizes Micropipettes comes in different sizes, each crafted to handle a specific range of volumes (Figure 1). For example, a P-10 micropipette handles volumes between 0.5 μL- 10 μL whereas a P-200 handles 20 μL- 200 μL. Every micropipette MUST be used together their corresponding, plastic- disposable tips. Handling solutions without these tips will contaminate/damage the micropipette. The volume range for some micropipettes overlap, such as the P-10 (0.5 μL- 10 μL) and the P-20 (2 μL- 20 μL). When pipetting volumes within this overlapping range, use the smallest micropipette which can handle the volume in one go. For example, use a P-10 to pipette 5 μL, and not P-20. Figure 1. Micropipettes. a) Two "1000 μL" pipettes from different manufacturers with major features labelled. The volume indicators are on their sides and can not bee seen here. For some pipettes (such as the Rainins), the plunger also acts as the volume adjustment knob. b) Different sizes of Rainin pipettes shown with their corresponding tips. Traditionally, pipettes for ~10 μL, ~100 μL and ~1000 μL are colour coded red, green and blue, respectively. BIO206 Fall 2024 / University of Toronto Mississauga 2 Volume indicators The volume indicator shows the volume of solution which the micropipettor is set to handle (Figure 1a). Reading these may be confusing at first since the digits of volume indicators are different for differently sized micropipettes. Use Figure 2 as a reference while learning how to read volume indicators. You don't need to memorize all of this at once. With some experience, this becomes second nature. The volumes of these micropipettes can be adjusted by simply turning their volume adjustment knobs. Some micropipettes such as the Rainins have a locking mechanism which needs to be disengaged before adjusting their volumes (the 'locked' and 'unlocked' positions beside the plunger can be seen in Figure 1b). Don't forget to lock it again before use. All volume-adjustable micropipettes have a minimum and a maximum volume which they can handle. NEVER adjust their volume knobs past this range as this may cause damage. Figure 2. Examples of volume indicators for typical micropipettes. Minimum and maximum volumes for each micropipette is shown alongside examples in between. BIO206 Fall 2024 / University of Toronto Mississauga 3 'First stop' and the 'Second stop' of the micropipette plunger After setting the volume and putting on the pipette tip, hold the micropipette upright and press on the plunger with your thumb to uptake/dispense liquids. The plunger is operated in a two-step mechanism, corresponding to the two positions which the plunger stops moving while you gently press on it. Practice operating the plunger in mid-air before you handle liquids. 1. Get a P-1000 micropipette and put on the appropriate tip. 2. Set the P-1000 to 500 μL and hold it upright. 3. Using your thumb, press on the plunger gently. Eventually, you should feel a firm resistance and the plunger stops moving so long as you are only applying gentle force. This is the first stop. 4. Then, push harder on the plunger. The plunger should move past the first stop and goes all the way down. This is the second stop. Micropipette practice exercise Make sure you have practiced using the plunger before you proceed. Materials Coloured water 1.5 mL microcentrifuge tubes Procedure (done individually) 1. Obtain a new 1.5 mL microcentrifuge tube. 2. Follow the step-by-step instructions in the 'Using a micropipette' section (on next page: page 5) to pipette 500 μL of coloured water into the 1.5 mL tube. 3. IMPORTANT: Self-check if you have accurately pipetted 500 μL into the 1.5 mL tube using the graduation marks on the tube. You must confirm with your TA that you know how to use a pipette correctly before proceeding. 4. Once you are comfortable with the P-1000, practice pipetting 150 μL, 75 μL, 15 μL, 5 μL and 2 μL into the 1.5 mL tube using appropriate micropipettes. Pipette these volumes into the same 1.5 mL tube. Feel free to get a new tube if necessary. Cleaning up Throw away the 1.5 mL tube with its contents into regular waste. Leave the coloured water on bench. BIO206 Fall 2024 / University of Toronto Mississauga 4 Using a micropipette Micropipettes are fragile and expensive to replace. Please use with care and respect. Every micropipette has a minimum/maximum volume it can handle. NEVER force the volume knobs past limits. Always use micropipettes with a tip. Always hold micropipettes upright; don't hold them upside down. 1. Chose the appropriately sized micropipette for the volume you are handling. 2. Adjust the volume to desired amount. Make sure you unlock/lock the micropipette appropriately if your micropipette has a locking mechanism. 3. Obtain a box of the corresponding tip and firmly press the micropipette into one of the tips. Do not 'bang' on the tips while putting them on. This is unnecessary. 4. While the tip is in mid air, press the plunger to the first stop. Hold the plunger at the first stop, and while holding, immerse the tip into the solution (Figure 3). 5. Slowly release the plunger to its home position. Observe the tip as the solution gets drawn in. Keep the tip immersed in solution until the plunger returns to the home position completely. Releasing the plunger too quickly results in inaccurate pipetting such as air bubbles getting introduced into the pipette tip. 6. Withdraw the tip from the solution. 7. Aim the end of the tip into the container which you want to dispense the solution. If possible, place the end of the tip gently against the wall of the container. This makes the solution 'stick' to the container, making it easier to dispense everything. 8. Gently press the plunger to the first stop, which will dispense most of the solution. Then, press the plunger to the second stop to completely dispense the rest of the solution remaining in the tip. Hold the plunger at the second stop and pull the tip out of the container. 9. Return the plunger to the home position. 10. You can keep using the same pipette tip so long as you are pipetting the same solution into a clean container. Otherwise, eject the tip into the waste container using the tip ejector. Figure 3. Inserting pipette tips into solution. Try to hold the end of the pipette tip close to the surface of the solution. Avoid inserting the tip deeply into solution as this may cause some of the solution to stick on the surface of the tip. BIO206 Fall 2024 / University of Toronto Mississauga 5 Part A2: 'Reagent mixing' exercise The 1.5 mL microcentrifuge tube is commonly used to set up reactions in a molecular biology lab. The following exercise simulates setting up reactions in a 1.5 mL tube. When setting up these reactions, add the reagent with the largest volume first to the tube since it's usually easier to handle larger volumes of solution. Rest of the reagents can be added directly into the first solution. Reagents in 1.5 mL tubes can be mixed by gently flicking the tube with your fingers and/or using a vortex (Appendix B). After mixing, it is a good practice to centrifuge the tubes briefly using a microcentrifuge (Appendix C) to collect all solution to the bottom of the tube. You don't want drops of reagents remaining on the side of the tubes, not participating in the reaction. A scientific equipment is only as accurate as their users. You can't assume that you will pipette accurately just because you are using a well calibrated micropipette. Pay close attention and handle the equipment appropriately to bring out their maximum performance. Materials Solution I Solution II Solution III Solution IV 1.5 mL microcentrifuge tubes Procedure (done individually) 1. Obtain three, clean 1.5 mL microcentrifuge tubes and label each A, B, and C using a permanent marker. Label 1.5 mL tubes on their lids. It's easier to see the labels this way. 2. Add reagents to Tube A following Table 1. Make sure to use fresh tips for different reagents. Begin by adding the largest volume first. 3. After pipetting all reagents into Tube A, mix well by vortexing / flicking with your fingers. 4. Put Tube A in a tabletop mini-centrifuge in a BALANCED way and perform a short spin (Appendix C). This pulls down all liquids to the bottom of the tube. Balance tubes with your group members. 5. When made correctly, Tube A should contain 200 μL of solution. Double check this by setting a P-200 to 200 μL and pipetting the contents of Tube A. If Tube A contained exactly 200 μL, the pipette tip should fill without air bubbles at the end while leaving any solution in Tube A. Other results indicate pipetting error. Have your TA check your pipetting technique. 6. Repeat the exercise for Tubes B and C and double check pipette accuracy. Double checking pipetting accuracy using a micropipette is not always necessary. However, using a micropipette to 'measure' volumes of solutions is a fairly common trick used in labs. BIO206 Fall 2024 / University of Toronto Mississauga 6 Table 1. Reagents to add to Tubes A, B and C. Volumes to add (μL) Total volume (μL) Solution 1 2 3 4 Tube A 40 12 70 78 200 Tube B 13 30 7 0 50 Tube C 3 5 0 2 10 Cleaning up Throw away the 1.5 mL tube with its contents into regular waste. Leave reagents I - IV on bench. BIO206 Fall 2024 / University of Toronto Mississauga 7 Part B: Spectrophotometric Analysis of Nucleic Acids Absorbance spectrophotometry is commonly used to measure the quantity and quality of nucleic acids in solution (read Appendix D for more on spectrophotometry). For nucleic acids, the amount of ultraviolet (UV) radiation, set at 260 nm wavelength, absorbed by your sample is used to calculate its concentration (absorbance at 260 nm = optical density at 260 nm = OD 260). The relationship between OD260 and sample concentration varies for different types of nucleic acids. For double-stranded DNA (dsDNA), OD260 of 1 corresponds to 50 µg/mL, assuming that the solution was measured using a cuvette with a 1 cm path length. For single-stranded DNA (ssDNA) or RNA, OD260 of 1 corresponds to 40 µg/mL. Purity of samples can be assessed using absorbance measurements at other wavelengths. The ratio of OD260 and OD280 is commonly used for this purpose (OD260/OD280). Pure solutions of DNA or RNA have OD260/OD280 of 1.8 and 2.0, respectively. OD260/OD280 smaller than these values typically indicate contamination by proteins and/or aromatic compounds such as phenol; the bigger the difference is between the measured ratio and the theoretical value, the more contaminated the solution. In addition, absorbance at 230 nm (OD230) can be used to indicate contamination by urea as well as aromatic compounds. As an example, let's say you measured a dsDNA solution which gave the following results: OD260 = 0.70 OD280 = 0.39 Remember that for a dsDNA solution: OD260 of 1 = 50 µg/mL OD260/OD280 of 1.8 = pure solution of dsDNA Therefore, for your dsDNA solution: Sample concentration = (50 µg/mL)(0.70) = 35 µg/mL Sample purity = 0.70/0.39 = 1.79 = this is a very pure (but not perfect) solution of dsDNA, as 1.79 is very close to the theoretical value for dsDNA, 1.8 In the following exercise, you will analyze a DNA solution of unknown concentration using spectrophotometry. Note 1: You suspect that the concentration of this solution is too high to be measured directly, and therefore decide to serially dilute the original solution before analysis. You are diluting the original solution to a final dilution factor of 200x (Figure 4). Note 2: We are using the Nanodrop spectrophotometer to analyze these DNA solutions (Figure 5). The Nanodrop allows us to analyze samples using a much smaller volume compared to a regular spectrophotometer. It also automatically calculates the concentration/purity of your DNA solution, but you are still required to be able to do these calculations manually. BIO206 Fall 2024 / University of Toronto Mississauga 8 Figure 4. Schematic diagram for diluting salmon sperm DNA. Take note that the third dilution step is a 2x dilution, not 10x. Figure 5. Small-volume spectrophotometers. a) Thermo Scientific NanoDrop 8000 spectrophotometer. b) Mettler Toledo UV5 Nano spectrophotometer. BIO206 Fall 2024 / University of Toronto Mississauga 9 Reagent handling techniques recap Use fresh tips when pipetting a different reagent. Mix reagents well after combining different reagents together. Once done mixing reagents, centrifuge the 1.5 mL microcentrifuge tubes to collect its contents at the bottom of tube. Materials Salmon sperm DNA solution (concentration unknown) deionized water 1.5 mL microcentrifuge tubes Nanodrop spectrophotometer (or equivalent) Procedure (done individually) 1. Obtain a clean 1.5 mL microcentrifuge tube and label it 'H 2O'. Transfer 1.0 mL of deionized water to this tube. Use this aliquot of water to set up your dilutions. Using smaller aliquots of reagents (in this case, water) to set up reactions reduces the numbers of times you open the larger storage container, lowering chance of contamination. 2. Plan your serial dilution for the original DNA solution by completing Table 2. A schematic of this dilution is shown in Figure 4. The total volume at each step of dilution should be 100 µL, and the final concentration relative to the original should be 200x. Show your calculation to your TA before you proceed. 3. Set up your tubes for serial dilution. Obtain three, clean 1.5 mL tubes and label them appropriately for the three dilution steps. Then, pipette the required volume of your diluent into each tube. 4. Start your serial dilution. Perform the first dilution by pipetting the required volume of your original DNA solution into the first tube. Mix very well and centrifuge briefly (no more than 3 - 4 seconds, using the 'short' spin function; Appendix C) to collect all solutions to the bottom of the tube by centrifugation. 5. Perform the second dilution by pipetting the required volume of your diluted DNA (in your first tube) into the second tube. Mix and centrifuge. 6. Perform the third dilution by pipetting the required volume of your diluted DNA (in your second tube) into the third tube. Mix and centrifuge. Your dilution is complete. 7. Analyze your third dilution using the Nanodrop spectrophotometer (or an equivalent equipment). Your TA will assist you in this process. 8. Record the concentration and purity of your diluted salmon sperm DNA solution. Remember, this is not the concentration of the original DNA solution given to you. 9. Calculate the concentration of your original salmon sperm DNA solution using the dilution factor of the third dilution relative to the original, stock DNA solution. BIO206 Fall 2024 / University of Toronto Mississauga 10 Table 2. Serial dilutions for salmon sperm DNA solution. The first dilution step is a 10x dilution of the original DNA solution. The second dilution step is a 10x dilution of the first dilution. The third dilution step is a 2x dilution of the second dilution. Volume of DNA Volume of Total volume Dilution factor sample to add diluent to add after mixing (μL) relative to (μL) (μL) original solution Tube 1 100 10x (dilution step 1) Tube 2 100 100x (dilution step 2) Tube 3 100 200x (dilution step 3) Cleaning up Throw away all tubes with its contents into regular waste (including original DNA solution, dilutions, etc.). Keep deionized water on benchtop. BIO206 Fall 2024 / University of Toronto Mississauga 11 Part C: Spectrophotometric Analysis of Proteins (Read-only) Concentrations of proteins in solution can also be determined by spectroscopy. Here, we will use the protein Bovine Serum Albumin (BSA) as an example. UV light between 270 nm - 280 nm is absorbed by aromatic amino acids tyrosine, tryptophan and phenylalanine. A simple way to determine concentration of a protein solution is to measure the sample between 250 nm to 310 nm. This typically produces a bell-shaped absorbance curve with the maximum absorbance (AbsMAX) occurring at around 275 nm (Figure 6). For BSA in solution measured using a 1 cm path length cuvette, absorbance of 1 at Abs MAX = 1 mg/mL of BSA. Figure 6. Spectrophotometric analysis of BSA using the BIO-RAD SmartSpec Plus Spectrophotometer. BSA solution was measured between 250 nm - 310 nm wavelength. For this sample, AbsMAX occurred at 272 nm with absorbance of 0.129 Absorbance Units. BSA concentration = (0.129)(1 mg/mL) = 0.129 mg/mL Different proteins absorb UV light with different rates as each protein has different amino acid compositions. Proteins with a higher proportion of aromatic amino acids will absorb UV light more effectively per molecule compared to proteins with a lower proportions of these amino acids. Thus, this method of measuring protein concentration is limited to proteins which their relationship between absorbance at ODMAX and their concentration is already known. BIO206 Fall 2024 / University of Toronto Mississauga 12 Appendix A: Metric units Table A1. Metric units and prefixes. a) Commonly used units for various measurements. b) Standardized prefixes assigned to an extremely wide range of numbers. Different prefixes are commonly used in various aspects of molecular biology. For example, sizes of genomes are typically in the giga and mega base-pairs. Concentrations and volumes of reagents are commonly within milli and micro range. Sizes of macromolecules are in the nano and pico range. a) b) Decimal Fractions Symbo Facto Fraction Arithmetic Symbol Term (prefix) Letter Abbreviation (prefix) Prefix l r Thousandth 10-3 milli m yotta Y 1024 Millionth 10-6 micro µ zetta Z 1021 Billionth 10-9 nano n exa E 1018 Trillionth 10-12 pico p peta P 1015 Molar Concentrations tera T 1012 Mole Fraction Name Letter Abbreviation Amount per Liter giga G 109 1M molar M 1 mole mega M 106 10-3 M millimolar mM 1 millimole kilo k 103 10-6 M micromolar µM 1 micromole hecto h 102 10-9 M nanomolar nM 1 nanomole deka da 101 Mass deci d 10-1 Name Abbreviation Fraction of a gram centi c 10-2 kilogram kg 103 milli m 10-3 gram g 1 micro u 10-6 milligram mg 10-3 nano n 10-9 microgram µg 10-6 nanogram ng 10-9 pico p 10-12 picogram pg 10-12 femt o f 10-15 Volume atto a 10-18 Name Abbreviation Fraction of a Liter zepto z 10-21 Liter L 1 milliliter mL 10-3 yocto y 10-24 microliter µL 10-6 Length Name Abbreviation Fraction of a meter Kilometer km 103 Meter m 1 Centimeter cm 10-2 Millimeter mm 10-3 Micrometer µm 10-6 Nanometer nm 10-9 Angstrom (not metric) Å 10-10 BIO206 Fall 2024 / University of Toronto Mississauga 13 Appendix B: Vortex A vortex is used to mix solutions vigorously (Figure A1). It's operation is simple: 1. Flip the selector to "ON" for continuous motion. Flip the selector to "Touch" to turn on the vortex only when samples are pressed onto the shaking platform. 2. Speed dial to set speed of vortex. 3. Shaking platform. Press samples onto this platform to activate vortex when it is set to "Touch" mode. Figure A1. A benchtop vortex. Numbers correspond to description in the text above. BIO206 Fall 2024 / University of Toronto Mississauga 14 Appendix C: Microcentrifuge A benchtop microcentrifuge, which the '1.5 mL microcentrifuge tubes' are named after, are used to centrifuge 1.5 mL tubes. This can be used to pellet large particles inside the tube and are also useful to collect solutions at the bottom of tube after mixing. A benchtop microcentrifuge does not produce as much centrifugal force as larger floor centrifuges and are relatively safe to operate (mishandling larger centrifuges could lead to lethal accidents). However, the smaller centrifuges still should be handled with care (Figure A2). 1. Press the 'Open' button to open the lid. Remove the cover of the rotor and place samples in a balanced configuration. You MUST place the rotor cover back in place. Close the lid. 2. Use time dial to set the duration of centrifugation. 3. Use speed dial to set the speed of centrifugation. 4. Use the rpm/rcf dial to select the unit of centrifugal force (or, the 'speed'). RPM = Rotations Per Minute. This sets the centrifugal force by choosing how many times the rotor rotates per minute. RCF = Relative Centrifugal Field. This sets the centrifugal force by choosing the amount of 'extra weight' used to pull samples towards the circumference of the rotor, relative to the gravity of the earth (1 g). For example, rcf of 1000x g means that samples will experience a pulling force 1000 times the force of gravity. 5. Press the start/stop button to start the centrifuge. Press it again to stop it prematurely in case of emergency (such as hearing unexpected noises, or seeing the centrifuge shake and move). Alternatively, press and hold the 'short' button to make the rotor spin at max speed. The rotor will spin as long as you are holding the button. This function is useful for brief spins to 'pull down solutions to the bottom'. Figure A2. Benchtop microcentrifuge. a) This microcentrifuge is set to spin samples at 14000 rpm for 3 minutes. b) The microcentrifuge rotor with its cover removed. This rotor has 18 spots to put in 1.5 mL microcentrifuge tubes. In this picture, two tubes are placed in a balanced configuration (assuming that these tubes contain equal amount of solution). BIO206 Fall 2024 / University of Toronto Mississauga 15 Appendix D: Spectrophotometry Many compounds absorb ultraviolet or visible light. Colorimetry measures the amount of light absorbed as it passes through the colored solution, and turbidimetry measures light absorption by particles in suspended in a solution. Colorimetry and Turbidimetry can be used to determine the concentration of colored chemicals and particles in suspension, respectively. The light absorbed by a colored solution (the optical density, or OD) or by particulate suspension is proportional to the concentration of the light absorbing material (Beers-Lambert Law). Photoelectric colorimeters and spectrophotometers are invaluable tools in the laboratory. A highly simplified diagrammatic representation of the photoelectric colorimeter is shown in Figure A3. The light from the lamp passes through a filter. The filter is determined by the properties of the solution being measured. The filtered monochromatic light passes through a cuvette containing the “blank” solution (100% transmission = 0% absorbance) or the sample to be measured. The light transmitted through strikes a photo cell that generates a current proportional to the intensity of the transmitted light. In the diagram, a beam of monochromatic light, of radiant power P0, directed at the sample solution is transmitted through the solution and now has radiant power P. Thus, light absorption has taken place in the solution. Millimeter or Galvanometer, combined with resistors and transformers to give P0 P measurement Light source Monochromatic Photocell filter Slit Slit Sample or Blank Figure A3. A simplified, schematic diagram of a photoelectric colorimeter. The amount of radiation absorbed may be measured in a number of ways: Transmittance, T = P / P0 % Transmittance, % T = 100 (T) Absorbance, OD = log10 P0 / P OD = log10 1 / T OD = log10 100 / % T OD = 2 - log10 % T [also stated as: %T = antilog (2.00 – OD)] BIO206 Fall 2024 / University of Toronto Mississauga 16 This equation, OD = 2 - log10 % T, is worth noting because it allows you to easily calculate absorbance (optical density) from percentage transmittance data. Percent transmittance plotted against concentration produces a curved line. However, plotting optical density against concentration gives a straight line (theoretically). This allows inferences about the concentration of the unknown solution by comparing with a single standard solution. Concentration of Unknown = OD of unknown * concentration of standard / OD of standard At high concentrations, solutions depart from this linear relationship. Therefore at high concentrations (OD > 1) should be diluted. BIO206 Fall 2024 / University of Toronto Mississauga 17 Appendix E: BIO-RAD SmartSpec Plus Spectrophotometer A benchtop spectrophotometer is a standard instrument in molecular biology labs due to its usefulness and ease of operation (Figure A4). The intensities of light-absorbing molecules can be measured directly which gives an insight to the concentration of those samples. Note that the 'colours' absorbed by of these molecules are not always visible light. DNA and proteins absorb light in the ultraviolet range. Also, 'colour-absorbing molecules' do not need to be macromolecules. For example, number of bacterial cells in a liquid culture can be determined by measuring absorbance of the culture at around 600 nm (discussed further in Lab 3). Figure A4. BIO-RAD SmartSpec Plus Spectrophotometer. A quick-start guide is available, pulled out from the bottom of the instrument. BIO206 Fall 2024 / University of Toronto Mississauga 18