Introduction to Spectrophotometry Objective: To learn how to use a spectrophotometer. Materials: UV/visible spectrophotometer Quartz Cuvette Polystyrene cuvettes Background Biophotonics is the study of the interaction of biological materials with light and other forms of radiant energy. Radiation is energy that comes from a source and can travel through material or space. The following illustration is of the electromagnetic spectrum along with various wavelengths. 1 Tissue and cells are composed of a variety of organic compounds (DNA, proteins, lipids, and carbohydrates). Light interacts with these molecules in different ways: reflection, absorption, transmission, and scattering. All biophotonic applications involve a light source that is passed through a target material and a detection sensor that reads the light emission from the material. A spectrophotometer has a light source that generates specific wavelengths. The light path passes through the cuvette, is absorbed by the material in the cuvette, and is read by a detector. The wavelength which the spectrophotometer is set to is referred to as the wavelength of absorbance. For example, a bacterium absorbs light at a wavelength of 600nm (A600). The amount of absorbed light per thickness of the sample is referred to as the Optical Density (OD). There are several different types of spectrophotometers available. We will use a UV/Visible spectrophotometer to measure the concentration of organic compounds. More specifically, we will measure the concentration of bacteria and DNA. UV/visible Spectroscopy are based on the absorption of light. This spectrophotometer uses a Xenon flash lamp which emits both UV and visible wavelengths. The path of light in the spectrophotometer is as follows: 2 Part I – Measuring bacterial growth Monitoring bacterial growth and harvesting a culture at the right moment is an important first step for any experiment which requires bacteria as a model organism. Bacterial growth occurs in four stages. The first phase, the lag phase, begins when the bacteria is added to the media. During this phase, bacterial growth is slow as the bacteria acclimate to the nutrients and the new environment. The second phase is called the log or exponential phase. It is during this phase in which the bacteria are reproducing at their maximum rate. The third phase is called the saturation or stationary phase. The growth rate of the bacteria slows during this phase due to the depletion of nutrients and the accumulation of waste products. The fourth and final phase is called the death phase. It is at this point that the bacteria begin to run out of nutrients and die. There are several techniques which can be utilized in order to measure bacterial growth. The first technique involves using a Hemocytometer. A Hemocytometer is used to count the number of cells in a given volume. The problem with using a Hemocytometer is that the total number of bacteria is determined not the number of bacteria which are alive. The second method used to determine bacterial concentration is serial dilution. A cell culture is diluted several times and plated. The number of colonies and the dilution factor are used in order to determine the concentration of the bacteria. The third technique involves using a spectrophotometer. The amount of light absorbed by the bacterial culture is measured. A wavelength of 600nm (A600) is used for measuring bacterial concentration. The benefit of using a spectrophotometer is that it’s quick and easy. The harvesting of a culture should be completed during the early log phase of cell growth. When measuring the rate of growth of bacteria in culture, an OD of .5-.7 indicates that the bacteria are in the early to mid log phase of growth. It is best to harvest the cells during this phase since the bacteria are at their peak rate of growth. 3 Protocol: Day 1: 1. Obtain three culture tubes. 2. Hypothesize how much media is necessary for each tube. 3. Add media to each tube. Be certain to record how much media has been added to each tube in the table below. 4. Add 5ul of DH5alpha cells to each culture tube. Record this amount in the data table 5. Incubate overnight at 37°C with vigorous shaking. 6. Remove the cultures from the incubator the following morning and place them in the refrigerator. Sample Volume of media Volume of DH5alpha cells OD600 reading 1 2 3 4 Day 2 1. Remove your cultures from the incubator/refrigerator. 2. Obtain four 1.5ml polystyrene cuvettes from your instructor. BE SURE TO HOLD THE CUVETTES ON THE RIGGED SIDES. FINGERPRINTS ON THE SMOOTH SIDES WILL DISTORT THE SPECTROPHOTOMETER READINGS. 3. Add 1ml of each overnight culture to a polystyrene cuvette. 4. Add 1ml of fresh media to the fourth cuvette (Used to zero the spectrophotometer) 5. Turn on the spectrophotometer. 6. Choose the bacteria concentration assay by pressing the OD600 button. 7. Press the “Enter” button for “Yes” when the screen prompts “A600 1.0=5.0e8cells/ml” 8. Place the cuvette with the fresh media into the sampling chamber. 9. Press the “Read Blank” button. 10. Remove the cuvette from the sampling chamber, and press the right arrow key to continue. 11. Place your first cuvette with culture into the sampling chamber and press the “Read Sample” button. 12. Record the OD600 reading and concentration in your lab notebook. 13. Remove the first sample and place the second sample into the sampling chamber. Press the “Read Sample” button. 14. Record the OD600 reading and concentration in your lab notebook. 15. When complete press the “Enter” key to return to the main screen. 16. Press the left arrow button to exit the assay. 5 Analysis: 1. Based on your OD readings approximately which phase of growth were your samples in? (0.5-0.7 indicates early to mid log phase of growth) 2. If you were to complete this experiment again what conditions would you change in order to better achieve an OD600 of 0.5-0.7? 6 Part II – Measuring DNA Concentration and Purity Both the purity and concentration may contribute to the success or failure of an experiment such as a bacterial transformation or mammalian cell transfection. For this experiment you will first determine the purity of a sample of DNA and then determine the sample concentration (ug/ml). A purity ratio is often used to determine the relative purity of a sample of DNA or RNA. DNA and RNA both absorb at a wavelength of 260nm while contaminants in the sample, such as proteins, absorb at a wavelength of 280nm. A ratio of the readings at A260 and A280 provides information on the relative purity of the DNA sample. An A260/A280 reading of 1.8 indicates a pure DNA sample while a reading of 2.0 indicates a pure RNA sample. The concentration of a DNA or RNA sample can also be estimated using a spectrophotometer. The following calculation can be used to determine the concentration of a sample of DNA or RNA Concentration = A260 * Conversion Factor * Dilution Factor The following conversion factors should be used when using this formula: Sample dsDNA ssDNA RNA A260 of 1.0 OD 50.0 ug/ml 37.0 ug/ml 40.0 ug/ml It is important to use a quartz cuvette when measuring RNA or DNA concentrations. Glass and plastic cuvettes absorb UV wavelengths, which limits their usefulness. 7 Protocol: 1. Complete a mini-prep on a bacterial culture in order to purify plasmid DNA.. 2. Obtain 2 microcentrifuge tubes 3. Fill one culture tube with 1ml of sterile water 4. In the other microcentrifuge tube, complete a 1:100 dilution of the stock DNA in order to obtain a final volume of 1ml. Vortex briefly to mix. 5. Turn on the spectrophotometer. 6. Choose the DNA/RNA assay. 7. Press the enter button to select dsDNA 8. Press the enter button when the screen reds “A260 1.0=50.0 ug/ml” 9. Obtain a quartz cuvette from the instructor. BE EXTREMELY CAREFUL WHEN HANDLING THE QUARTZ CUVETTE! AVOID FINGERPRINTS ON THE SURFACE! DO NOT DROP THE CUVETTE! 10. Press the dilution factor button and set the dilution factor to 100. Press enter. 11. Zero (blank) the spectrophotometer by placing 1ml of water in the cuvette from the first culture tube. 12. Place the cuvette into the sample chamber and press the “Read Blank” button. 13. Empty out the quartz cuvette and place 1ml of your diluted DNA sample into the cuvette. 14. Place the cuvette into the sample chamber and press the “Read Sample” button. 15. Collect the absorbance data for A260. Record this information in the data table below. 16. Press the “Abs” key to collect the A280 data. Record this information in the data table below. 8 17. Collect the concentration data for A280 and A260 and record the information in the data table below. 18. Press the “A260:A280” button and record the purity ratio in the table below. 19. When complete press the “Enter” key to return to the main screen. 20. Press the left arrow button to exit the assay. Sample Dilution Factor A280 (OD) A260 (OD) concentration (ug/ml) A260:A280 Analysis: 1. What is the concentration of your DNA sample (ug/ul)? A DNA mini prep can yield up to .1ug/ul of DNA, how does your sample compare? 2. What is the purity ratio of your sample? Considering that a sample of pure DNA has a purity ratio of 1.8, how pure is your sample? 9