Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants

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1 The Biotechnology Education Company Revised and Updated Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Storage: See Page 3 for specific storage instructions Experiment Objective: This exercise simulates detection of multiple water microorganisms using PCR to simultaneously amplify DNA from three different bacterial strains present in water This experiment is designed for DNA staining with InstaStain Ethidium Bromide. EDVOTEK, Inc EDVOTEK

2 2 xxx Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Table of Contents Experiment Components 3 Experiment Requirements 4 Background Information 5 Experiment Procedures Experiment Overview and General Instructions 9 Laboratory Safety 10 Module I: Isolation of DNA 11 Module II: PCR Amplification of the Multiplex Water Contaminants 12 Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis 14 Study Questions 16 Instructor's Guidelines Notes to the Instructor 18 Pre-Lab Preparations 21 Experiment Results and Analysis 23 Study Questions and Answers 24 Appendices A PCR Experimental Success Guidelines 26 B Polymerase Chain Reaction Using Three Water baths 28 C Preparation and Handling of PCR Samples With Wax 29 D 1.0% Agarose Gel Preparation 30 E 1.0% Agarose Gels - Quantity Preparations 31 F Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards 32 G InstaStain Methylene Blue: One Step Staining and Destaining 33 Material Safety Data Sheets 34 EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc. EDVOTEK -

3 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 3 Components & Requirements This experiment is designed for 20 students working in groups of five. Storage A Tubes with PCR reaction pellets Room Temperature Each PCR reaction pellet contains dntp Mixture Taq DNA Polymerase Buffer Taq DNA Polymerase MgCl 2 B Primer mixture -20 C Freezer (contains E. coli-specific primers, Bacillus subtilis-specific primers, and Serratia marcescens-specific primers C 200 base pair ladder -20 C Freezer D E. coli DNA for positive control -20 C Freezer E Tris buffer -20 C Freezer F Proteinase K Room temperature G Potassium Acetate Room temperature H DNA Extraction buffer Room temperature E. coli culture Room temp. desiccated Bacillus subtilis culture Room temp. desiccated Serratia marcescens culture Room temp. desiccated Reagents & Supplies (Store all components below at room temperature) All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. of the experiment components are derived from human sources. UltraSpec-Agarose Electrophoresis Buffer (50x) 10x Gel Loading Solution InstaStain Ethidium Bromide InstaStain Methylene Blue Microcentrifuge Tubes PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template) 50 ml Conical tube Wax beads (for water bath option or thermal cyclers without heated lid) EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

4 4 xxx Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Requirements *If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three water baths. However, a thermal cycler assures a significantly higher rate of success. Thermal cycler (EDVOTEK Cat. # 532 highly recommended) or three water baths* Horizontal gel electrophoresis apparatus D.C. power supply Balance Microcentrifuge Water bath (70 C) UV Transilluminator or UV Photodocumentation system UV safety goggles Automatic micropipets (5-50 µl) with tips Microwave, hot plate or burner Pipet pump 250 ml flasks or beakers Hot gloves Disposable vinyl or latex laboratory gloves Ice buckets and ice Distilled or deionized water Online Ordering now available Visit our web site for information about EDVOTEK s complete line of hands-on experiments for biotechnology and biology education. EDVO-TECH SERVICE EDVOTEK Mon - Fri ( ) 9 am - 6 pm ET Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) Web: edvotek@aol.com Please have the following information ready: Experiment number and title Kit lot number on box or tube Literature version number (in lower right corner) Approximate purchase date EDVOTEK -

5 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 5 Multiplex PCR Analysis for Water Contaminants Contaminated drinking water is the leading cause of infectious disease in the world, resulting in over nine million deaths each year. Water pollution that is direct, from a single, readily identifiable source, such as a manufacturing plant or water treatment facility (Figure 1), is known as point source and is strictly regulated by government agencies. Nonpoint source pollution, by contrast, is that which is not attributable to a single, identifiable source, and involves polluted water runoff resulting from everyday activities. Typical nonpoint sources include discharge from septic tanks, fertilizer runoff from farm areas, forest soil erosion, and chemical spills. Such runoff can contaminate rivers, lakes, and streams and eventually proceed to taint drinking water supplies. As nonpoint pollution results primarily from the actions of individuals, mindfulness of the impact of dumping garbage, oil, etc., can help lessen its harmful consequences. Background Information Figure 1. Point and Nonpoint sources of water contamination.

6 6 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Multiplex PCR Analysis for Water Contaminants Background Information As both point and nonpoint pollution eventually contaminate water sources used for drinking water, water treatment must effectively remove these impurities. While drinking water in U.S. cities is generally safe, several outbreaks still occur each year, making monitoring still necessary. Additionally, private well owners should also test water regularly for harmful chemicals, algae, and bacteria. Waterborne microbes such as Giardia and Cryptosporidium can cause severe, life-threatening illness if ingested in sufficient quantities (these are not actually bacteria but single-celled organisms called protists). Giardia and Cryptosporidium exist in the environment as cysts, hard, biologically inert spheres that begin to divide when ingested. Similarly, Salmonella and Shigella are two other (bacterial) waterborne pathogens that can cause community-wide infectious disease. In particular, elderly people or those afflicted with AIDS, cancer, and other diseases can have weakened immune systems that make them more vulnerable to such infections. Because testing for multiple organisms is time-consuming and prohibitively expensive, water is typically analyzed for the presence of a group of relatively harmless bacteria, known as coliform. Coliform bacteria are often used to indicate the presence of more harmful organisms and include bacteria such as Escherichia Coli (E. Coli), Klebsiella, Enterobacter, and Serratia. These organisms normally inhabit the intestine and gut of animals and humans. If a water sample is negative for coliform bacteria, it is assumed that more harmful organisms are also absent, and the water is deemed safe for drinking or swimming. In addition to the coliform test, a separate second test is often performed for E. Coli, whose presence could indicate fecal contamination. If a sample tests positive for E. Coli or coliform, it is often sent to a wellequipped laboratory that can perform more complex (and more expensive) tests to determine the specific (possibly pathogenic) organisms that might be present. One laboratory method that is now universally used to detect specific microorganisms is the polymerase chain reaction (PCR). PCR, invented in 1986 by Dr. Kary Mullis at Cetus Corporation (for which he later received a Nobel Prize), has become invaluable for the amplification of specific DNA sequences. The basis for PCR is a heat-stable DNA polymerase (usually Taq polymerase) that can withstand near-boiling temperatures. Applications of PCR include genetic disease diagnosis, forensic identification, paternity testing, and detection of specific microorganisms (usually bacteria and viruses) in body fluids, soil, and water.

7 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 7 Multiplex PCR Analysis for Water Contaminants The basic PCR reaction is shown in Figure 2. The first step involves denaturation of the DNA sample to be tested (e.g., an unknown water sample). Denaturation involves separation of the two DNA strands at near boiling, generally around 95 C, at which the Taq polymerase remains stable. The temperature is then lowered to 45 to 60 to allow binding (annealing) of sequence-specific primers, short DNA molecules (Figure 1, small red and blue bars), to the source DNA (template). The primers are designed to bind only to the sequence to be amplified, called the target sequence (Figure 1, light green bar). An example of a target PCR sequence would be a gene found only in a specific water bacterium. Following primer annealing during the second PCR step, the temperature is raised to 72 C. At this temperature, the polymerase can add new bases to the primers in a process known as extension, creating new DNA molecules between each primer-binding site. These three steps: denaturation, annealing, and extension, together constitute one PCR cycle. The entire process is then repeated, resulting in a doubling of the target sequence following each cycle. The PCR reaction is generally repeated for cycles, resulting in an ideal amplification of fold of the original DNA target; in reality, however, the exact amplification is lower, due to imperfect reaction conditions. Background Information For water microbe detection, PCR can be used to sensitively and exclusively detect specific aquatic organisms. For example, given a positive result for total coliform, one may further test the water sample, using PCR, for pathogenic organisms such as Cryptosporidium. For specific detection, primers are designed to correspond to gene sequences distinct to those species. In this experiment, PCR tests will be performed to concurrently detect specific bacteria, including E. coli, Bacillus subtilis, and Serratia marcescens. A multiplex PCR reaction is one in which multiple amplifications are performed simultaneously using several primer pairs in a single tube. This exercise will simulate follow-up testing that occurs subsequent to a positive coliform test. The experiment will thus demonstrate concurrent detection of specific water pathogens, using specific primers for unique, organism-specific genes, in a single PCR reaction.

8 8 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Multiplex PCR Analysis for Water Contaminants Target Sequence = Separation of two DNA strands 3' 3' = Primer 1 The Experiment Cycle 1 = Primer 2 3' 3' 3' 3' Denature 94 C Anneal 2 primers 40 C - 65 C 3' 3' Extension 72 C Cycle 2 3' 3' 3' 3' 3' 3' Cycle 3 3' 3' 3' 3' 3' 3' 3' 3'

9 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 9 Experiment Overview and General Instructions BEFORE YOU START THE EXPERIMENT 1. Read all instructions before starting the experiment. 2. If you will be conducting PCR using a thermal cycler without a heated lid, also read the Appendix entitled Preparation and Handling PCR Samples with Wax. If you will be using three water baths to conduct PCR, read the two appendices entitled Polymerase Chain Reaction Using Three Water baths and Handling samples with wax overlays. 3. Write a hypothesis that reflects the experiment and predict experimental outcomes. EXPERIMENT OBJECTIVE: The Experiment The objective of this experiment is to simulate the detection of multiple water microorganisms using PCR to simultaneously amplify DNA from three different bacterial strains present in water. Brief Description of Experiment: In this experiment, each student will isolate DNA from different bacterial cultures and perform PCR to differentiate between the organisms. Gel specifications This experiment requires a gel with the following specifications: Recommended gel size 7 x 14 cm (long tray) Number of sample wells required 6 Placement of well-former template first set of notches Gel concentration required 1.0%

10 10 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Laboratory Safety 1. Gloves and goggles should be worn routinely as good laboratory practice. The Experiment 2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. 3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS. Wear gloves and safety goggles 4. Exercise caution when using any electrical equipment in the laboratory. Although electrical current from the power source is automatically disrupted when the cover is removed from the apparatus, first turn off the power, then unplug the power source before disconnecting the leads and removing the cover. Turn off power and unplug the equipment when not in use. 5. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. However, in the unlikely event that a leak develops in any electrophoresis apparatus you are using, IM- MEDIATELY SHUT OFF POWER. Do not use the apparatus. 6. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

11 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 11 Module I: Isolation of DNA Work in groups of 5, with each group member working with a different water sample (Bacillus, E. Coli, Serratia, unknown #1, and unknown #2). WARNING! Use only screw-cap tubes when incubating in the water bath for DNA isolation. Do not use snap-top tubes. Isolation of bacterial DNA from five cultures 1. Obtain one of the five different bacteria in a 1.5 ml screw-cap microcentrifuge tube from your instructor. 2. Centrifuge for 5 minutes, at maximum speed, in a balanced microcentrifuge to pellet the cells. 3. Remove and discard the supernatant - use care not to disturb the pellet. 4. Mix the lysis buffer well. Add 100 µl lysis buffer to the pellet. 5. Vortex or tap vigorously to resuspend the pellet. 6. Float tubes in a 70 C water bath for 15 minutes. 7. Add 14 µl of potassium acetate to each tube and mix for 5 seconds. Leave on ice for 5 minutes. The Experiment After centrifugation, mark location of the pellet 8. Spin the tubes in a microcentrifuge at maximum speed for 5 minutes. 9. Carefully transfer just the supernatant into a fresh, labeled 0.5 ml microcentrifuge tube, being careful not to disturb the pellet. (Discard the tube with the pellet after the supernatant is saved). 10. Precipitate the DNA in the supernatant by adding 45 µl of room temperature isopropanol to each tube. Use a marker to draw a circle at the bottoms of the tubes so you can locate the DNA pellet after centrifugation (see drawing at left). Spin at maximum speed for 5 minutes. 11. Carefully remove and discard all of the supernatant (avoid losing the DNA pellet). A very small DNA pellet should be visible at the bottom of the tube where it was marked in the previous step. WARNING: The DNA pellet is very small. Take care not to lose it! 12. Wash the pellet carefully with 20 µl of 70% ETOH. Spin the tube(s) at maximum speed for 3 minutes. 13. Discard the supernatant and allow the pellet to completely dry. Resuspend the DNA pellet in 25 µl of TE buffer. 14. Store the tubes on ice until you are ready to proceed with Module II, or freeze the tubes for future use. OPTIONAL STOPPING POINT Place bacterial lysate in -20 C freezer.

12 12 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Module II: PCR Amplification of the Multiplex Water Contaminants PCR Amplification of Bacterial DNA The Experiment The PCR reaction pellet contains Taq DNA polymerase, the four deoxytriphosphates, Mg +2 and buffer. Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for seconds at maximum speed. 1. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler. 2. Label the tube containing the PCR reaction pellet with the sample and your initials. 3. To the PCR Reaction pellets, add components as outlined in the table below. 4. Gently mix the PCR reaction tube and quickly spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet is completely dissolved. 5. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. If your thermal cycler does not have a heated lid, or if you are cycling manually with three water baths, add one wax bead to the tube before proceeding to polymerase chain reaction cycling. E B S Unk #1 Unk #2 Primer mixture 20 µl 20 µl 20 µl 20 µl 20 µl E. coli DNA 5 µl B. Subtilis DNA 5 µl S. marcescens DNA 5 µl Unknown #1 DNA 5 µl Unknown #2 DNA 5 µl Total 25 µl 25 µl 25 µl 25 µl 25 µl

13 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 13 Module II: PCR Amplification of the Multiplex Water Contaminants optional positive control 1. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler. Label the tube Positive control. 2. Add the following to the pellet: Primer solution 20 µl E. coli DNA 5 µl 3. Gently mix the tube and briefly spin it in a microcentrifuge to collect all of the sample at the bottom of the tube. Make sure the PCR pellet is completely dissolved. 4. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling. If your thermal cycler does not have a heated lid, or if you are cycling manually with three water baths, add one wax bead to the tube before proceeding to polymerase chain reaction cycling. The Experiment Polymerase Chain Reaction Cycling 1. For automatic cycling, each student should place his/her PCR tube (and the optional control reaction) in the programmed thermal cycler. Follow the same cycling schedule if you are manually cycling in three water baths. Initial Denaturation 35 Final Extension 94 C for 5 min. 94 C for 30 sec. 72 C for 4 min. 58 C for 30 sec. 72 C for 60 sec. 2. After the cycles are completed, add 5 µl of 10x Gel Loading Solution to the sample and store on ice until ready for electrophoresis. 3. Proceed to instructions for preparing a 1.0% agarose gel (7 x 14 cm) and separating the PCR products by electrophoresis. OPTIONAL STOPPING POINT The samples can be held in the thermal cycler at 4 C or frozen after addition of 5 µl of 10x Gel Loading Solution until ready for electrophoresis.

14 14 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis The Experiment If you are unfamiliar with agarose gel preparation and electrophoresis, detailed instructions and helpful resources are available at Agarose Gel Requirements Recommended gel size: 7 x 14 cm 7 x 14 cm gels are recommended to achieve better resolution of the PCR products. Each gel can be shared by several students or groups. Placement of well-former template: first set of notches Agarose gel concentration: 1.0% Preparing the AGAROSE GEL 1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape. 2. Place a well-former template (comb) in the first set of notches at the end of the bed. Make sure the comb sits firmly and evenly across the bed. Important Note Continue heating until the final solution appears clear (like water) without any undissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved. 3. To a 250 ml flask or beaker, add agarose powder and buffer as indicated in the Reference Tables (Appendix A) provided by your instructor. Swirl the mixture to disperse clumps of agarose powder. 4. With a marking pen, indicate the level of the solution volume on the outside of the flask. 5. Heat the mixture using a microwave oven or burner to dissolve the agarose powder. 6. Cool the agarose solution to 60 C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume marked in step 4. After the gel is cooled to 60 C: 7. Place the bed on a level surface and pour the cooled agarose solution into the bed. 8. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. 9. After the gel is solidified, be careful not to damage or tear the wells while removing the rubber dams or tape and comb(s) from the gel bed. 10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform. 11. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer (refer to Table B on the instruction Appendix provided by your instructor).

15 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 15 Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain Ethidium Bromide. Loading DNA Samples Reminder: Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. Black Sample wells + Red 1. Heat the 200 bp ladder and PCR samples for two minutes at 50 C. Allow the samples to cool for a few minutes. 2. Make sure the gel is completely submerged under buffer before loading the samples. Load the entire volume (30 µl) of the samples in the following sequence: Lane Tube 1 A 200 bp DNA ladder 2 B Positive PCR control 3 C B. subtilis 4 D E. Coli 5 E S. marcescens 6 F Unknown #1 or Unknown #2 The Experiment E. 3. Record the position of your sample in the gel for easy identification after staining. Running the Gel 4. After the DNA samples are loaded, properly orient the cover and carefully snap it onto the electrode terminals. 5. Insert the plugs of the black and red leads into the corresponding inputs of the power source. 6. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor. 7. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. 8. After the electrophoresis is completed, disconnect the power and remove the gel from the bed for staining. Staining and visualization of DNA After electrophoresis, agarose gels require staining to visualize the separated DNA samples. Your instructor will provide instructions for DNA staining with InstaStain Ethidium Bromide.

16 16 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. What is non-point water pollution? Why is it important to be aware of this type of pollution? The Experiment 2. What are some applications of PCR? How can it be used to monitor water contamination? 3. What is coliform? If a sample is positive for coliform, what action should be taken? 4. What is Giardia?

17 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 17 Instructor s Guide Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at EDVOTEK ( ). National Content and Skill Standards By performing this experiment, students will learn to load samples and run agarose gel electrophoresis. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation. Please visit our web site for specific content and skill standards for various experiments. EDVO-TECH SERVICE EDVOTEK Mon - Fri ( ) 9 am - 6 pm ET Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) Web: edvotek@aol.com Please have the following information ready: Experiment number and title Kit lot number on box or tube Literature version number (in lower right corner) Approximate purchase date Educational resources Electrophoresis Hints, Help and Frequently Asked Questions EDVOTEK experiments are easy to perform and designed for maximum success in the classroom setting. However, even the most experienced students and teachers occasionally encounter experimental problems or difficulties. The ED- VOTEK web site provides several suggestions and reminders for conducting electrophoresis, as well as answers to frequently asked electrophoresis questions. Online Ordering now available Visit our web site for information about EDVOTEK s complete line of hands-on experiments for biotechnology and biology education. EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

18 18 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Notes to the Instructor: PCR EXPERIMENTAL SUCCESS GUIDELINES Please refer to the Appendices section for a summary of important hints and reminders which will help maximize successful implementation of this experiment. This experiment has three modules: The Experiment I. Isolation of DNA II. PCR Amplification of Multiplex Water Contaminants III. Separation of PCR Reactions by Electrophoresis Micropipetting Basics and Practice Gel Loading Accurate pipeting is critical for maximizing successful experiment results. EDVOTEK Series 300 experiments are designed for students who have had previous experience with agarose gel electrophoresis and micropipeting techniques. If your students are unfamiliar with using micropipets, EDVOTEK highly recommends that students perform Experiment # S-44, Micropipetting Basics, or other Series 100 or 200 electrophoresis experiment prior to conducting this advanced level experiment. APPROXIMATE TIME REQUIREMENTS 1. The PCR steps will take about minutes or can be processed overnight and held at 4 C. 2. The experiment can be temporarily stopped after the completion of Modules I and II and later resumed. Experimental results will not be compromised if instructions are followed as noted under the heading Optional Stopping Point at the end of Module I and Module II. 3. Whether you choose to prepare the gel(s) in advance or have the students prepare their own, allow approximately minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification. See section Options for Preparing Agarose Gels below. Table C Volts Time and Voltage (1.0% - 7 x 14 cm gel) Recommended Time Minimum Maximum 55 min 2 hrs 15 min 3 hrs 25 min 1 hr 15 min 3 hrs 5 hrs 4. The approximate time for electrophoresis will vary from 1-5 hours. Generally, the higher the voltage applied, the faster the samples migrate. However, depending upon the apparatus configuration and the distance between the two electrodes, individual electrophoresis units will separate DNA at different rates. Follow manufacturer's recommendations. Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C.

19 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 19 Notes to the Instructor: options for Preparing AGAROSE gels This experiment is designed for DNA staining after electrophoresis with InstaStain Ethidium Bromide. There are several options for preparing agarose gels for the experiment. 1. Individual Gel Casting: Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment. 2. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be stored under buffer in the refrigerator for up to 2 weeks. Do not store gels at -20 C. Freezing will destroy the gels. Gels that have been removed from their trays for storage, should be "anchored" back to the tray with a few drops of hot, molten agarose before placing the gels into the apparatus for electrophoresis. This will prevent the gels from sliding around in the trays and the chambers. Instructor s Guide 3. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for sharing by the class. See instructions for "Batch Gel Preparation". Gel concentration and volume The gel concentration required for this experiment is 1.0%. Prepare gels according to Table A.1 or A.2 in Appendix D.

20 20 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Notes to the Instructor: Gel Staining and Destaining AFTER ELECTROPHORESIS Instructor s Guide After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain. InstaStain EtBr (Appendix F) Optimal visualization of PCR products on gels of 1.0% or higher concentration is obtained by staining with InstaStain Ethidium Bromide (InstaStain EtBr) cards. Exercise caution when using Ethidium Bromide, which is a listed mutagen. Disposal of the InstaStain EtBr cards, which contain only a few micrograms of ethidium bromide, is minimal compared to the large volume of liquid waste generated by traditional ethidium bromide staining procedures. Disposal of InstaStain cards and gels should follow institutional guidelines for chemical waste. InstaStain MetBlue: One-step Staining and Destaining (Appendix G) InstaStain Methylene Blue (InstaStain MetBlue) can be used as an alternative for staining gels in this experiment. However, InstaStain MetBlue is less sensitive than InstaStain EtBr and will yield variable results. Agarose gels can be stained and destained in one easy step, which can be completed in approximately 3 hours, or can be left in liquid overnight. For the best photographic results, leave the gel in liquid overnight. This will allow the stained gel to "equilibrate" in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background. Gels stained with InstaStain Methylene Blue may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS! Used InstaStain MetBlue cards and destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed down the drain. Photodocumentation of DNA (Optional) There are many different photodocumentation systems available, including digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using.

21 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 21 Pre-Lab Preparations 1. Prepare the contaminated water samples: Using a separate sterile loop for each culture, collect the cells by gently scraping the surface of each bacterial slant (B. subtilis, E. coli, S. marcescens). Draw the loop towards the top of the slant to collect the cells. Transfer each culture into separate, labeled 50 ml conical tubes each containing 40 ml of distilled or deionized water. Use caution to avoid cross-contamination. Mix the "contaminated water" for 15 minutes at room temperature. Alternatively, samples can be placed on a shaking platform. 2. Prepare the unknowns Combine 5 ml of the E. coli and 5 ml of the S. marcescens and label this "Unk #1". Prepare a mixture of any combination of the bacteria for unknown #2. Label this "Unk #2". Instructor s Guide 3. Label and dispense 1.5 ml of each of the samples into 1.5 ml screw-cap tubes for each of the lab groups. 4. Resuspend each of the two tubes of Proteinase K with 50 µl of DNA Extraction buffer (H). Allow the Proteinase K to rehydrate for a minute and mix completely to dissolve. Each student group of five will need the following: 5 Tubes, each containing one of the contaminated water samples (in screw-cap tubes) ml screw-cap tubes 1 Tube of Lysis Solution (0.6 ml), on ice ml snap-top tubes 5 Tubes, each containing a PCR pellet 1 Tube of multiplex primer solution (130 µl) 1 Tube of Gel loading solution (50 µl) 1 Tube of 200 bp ladder (35 µl) 1 Tube of Potassium acetate (100 µl) 1 Tube of Tris buffer (150 µl) 5. Add all of the dissolved Proteinase K back to the remaining DNA Extraction buffer. Mix well and label Lysis Solution. Mix the Lysis solution between each aliquot and dispense 0.6 ml of Lysis Solution for each group. Keep on ice. 6. Dispense 130 µl of primer mixture (B) into labeled tubes, one per group. Keep on ice. 7. Aliquot 35 µl of 200 bp DNA ladder (C) into labeled tubes, one per group. 8. Dispense 150 µl of Tris buffer (E) into labeled tubes, one per group. 9. Dispense 100 µl of Potassium acetate (G) into labeled tubes, one per group. 10. Prepare 70% ethanol and isopropanol. Keep on ice. Divide students into four groups of five. Each student can work independently on one of the contaminated water samples. Designate one group to use the positive control DNA (D) for the control PCR reaction.

22 22 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Pre-Lab Preparations Notes and Reminders: Instructor s Guide Accurate temperatures and cycle times are critical for PCR. A pre-run for one cycle (approx. 3 to 5 minutes) is recommended to check that the thermal cycler is properly programmed. For thermal cyclers that do not have a top heating plate, it is necessary to place a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix entitled "Preparation and Handling PCR Samples with Wax ". Three water baths can be used for PCR if a thermal cycler is unavailable. The experiment will require great care and patience. Samples will require wax layers. See appendices entitled "Polymerase Chain Reaction Using Three Water baths" and "Handling samples with wax overlays". MODULE II - AMPLIFICATION OF THE Multiplex Water Contaminants The Thermal cycler should be programmed for the following cycles: Initial Denaturation 35 Final Extension 94 C for 5 min. 94 C for 30 sec. 72 C for 4 min. 58 C for 30 sec. 72 C for 60 sec. Accurate temperatures and cycle times are critical. A pre-run for one cycle (which will take approximately 3 to 5 min) is recommended to check that the thermal cycler is properly programmed. For thermal cyclers which do not have a top heating plate, it is necessary to place a layer of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. Three water baths can be used for PCR if a thermal cycler is unavailable. The experiment will work if given great care and patience. Samples will require wax layers.

23 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 23 Experiment Results and Analysis The PCR products should have the following sizes: B. subtilis 893 bp E. Coli 479 bp S. marcescens 629 bp In this example, the positive PCR control sample is shown in Lane 2. Water samples contaminated with B. subtilis, E. Coli, and S. marcescens are shown in Lanes 3, 4 and 5, respectively. Lane 6 shows Unknown #1 contaminated with E. coli and S. marcescens. 200 bp ladder Positive PCR control B.subtilis E. coli S. marcescens Unknown #1 Instructor s Guide 1200 bp 1000 bp 800 bp 600 bp 400 bp 200 bp

24 24 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Study Questions and Answers 1. What is non-point water pollution? Why is it important to be aware of this type of pollution? Instructor s Guide Nonpoint water pollution refers to that which cannot be traced to a single source and originates from water washing over land and depositing contaminants into waterways. Nonpoint pollution sources include sediment from soil erosion, farmland fertilizer runoff, faulty septic systems, and spilled chemicals/waste. As this type of water contamination usually results from the conduct of individuals, avoidance of garbage dumping, etc., can greatly alleviate environmental harm resulting from such activities. 2. What are some applications of PCR? How can it be used to monitor water contamination? PCR is now universally used for genetic disease diagnosis, forensic identification, paternity testing, and detection of specific pathogens in water and food. For detection of water contamination with specific microorganisms, primers are designed for genes that are unique to those organisms. 3. What is coliform? If a sample is positive for coliform, what action should be taken? Coliform refers to a group of relatively harmless bacteria that normally inhabit the intestine and gut of mammals. Most water contamination tests assay for the presence of coliform bacteria; the presence of these could indicate the presence of more harmful microorganisms. 4. What is Giardia? Giardia is a pathogenic microorganism, called a protist, which infects the human small intestine. Giardia cysts are passed though animal feces; thus, drinking of fecal-contaminated water can result in a severe Giardia infection (called giardiasis). Giardiasis can cause severe diarrhea and other symptoms, especially in people with deficient immune systems (e.g., AIDS and cancer patients, elderly persons, etc.).

25 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants 25 Appendices A B C D E F G PCR Experimental Success Guidelines Polymerase Chain Reaction Using Three Water baths Preparation and Handling of PCR Samples With Wax 1.0% Agarose Gel Preparation 1.0% Agarose Gels - Quantity Preparations Staining and Visualization of DNA with InstaStain Ethidium Bromide Cards InstaStain Methylene Blue: One Step Staining and Destaining Material Safety Data Sheets EDVOTEK - The Biotechnology Education Company EDVOTEK FAX: (301) info@edvotek.com

26 26 Appendix A PCR Experimental Success Guidelines EDVOTEK experiments which involve the extraction and amplification of DNA for fingerprinting are extremely relevant, exciting and stimulating classroom laboratory activities. These experiments have been performed successfully in many classrooms across the country, but do require careful execution because of the small volumes used. The following guidelines offer some important suggestions, reminders and hints for maximizing success. Important Note Remember: Any carryover of chelating agent to the PCR reaction will not yield results. DNA Extraction and sample preparation: 1. Sufficient Cells: It is critical that there are sufficient cells to obtain enough DNA that will yield positive DNA fingerprinting results. Cell sources include human, plant, drosophila and bacterial cells. Without enough cells, there will not be enough DNA template for the PCR reaction. 2. Lysis Solution: The lysis solution contains chelating agent which removes Mg (required by DNA-degrading nucleases and DNA polymerases). The small beads must be suspended in the buffer prior to delivery to the cells (i.e., mix the lysis solution just before you transfer it to the tube containing the cells. 3. Boiling: The boiling step is required to obtain cell lysis. Boiling will not degrade the DNA and nucleases will NOT degrade DNA in the absence of Mg. 4. Centrifugation: Centrifuge the cell suspension carefully after cooling. If the pellet loosens, repeat this step. The supernatant should be clear, not cloudy, and the pellet should be solid at the bottom of the tube. Repeat centrifugation for a longer period of time, if necessary. 5. DNA Transfer: Transfer the DNA to a new microcentrifuge tube very carefully. It is the step prior to the PCR reaction. If any chelating agent beads (as few as one or two) are transferred, they can easily trap the Mg required by the Taq DNA polymerase as a cofactor for catalysis. As an additional precaution, centrifuge the supernatant a second time. The PCR Reaction 6. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward and reverse primers) and the cell DNA (supernatant) as specified in the experimental procedures to the microcentrifuge tube containing the PCR reaction bead. Make sure that the bead (which contains the Taq DNA polymerase, the 4XdTPs, Mg and the PCR reaction buffer) is completely dissolved. Do a quick spin in a microcentrifuge to bring the entire sample to the bottom of the tube. Prepare the control reaction similarly. 7. The Thermal cycler: The thermal cycler must be programmed for the correct cycle sequence. It is critical that the temperatures and the time for each of the cycles are accurate.

27 Appendix A PCR Experimental Success Guidelines (continued) Oil or Wax: For thermal cyclers that do not have a top heating plate, the reaction in the tubes must be overlaid with oil or wax to prevent evaporation. 9. Manual Water Bath PCR: Three water baths can be used as an alternative to a thermal cycler for PCR, but results are more variable. Samples require oil or wax layers. This method requires extra care and patience. Gel Preparation and Staining 10. Concentrated agarose: Gels of higher concentration (> 0.8%) require special attention when dissolving or re-melting. Make sure that the solution is completely clear of clumps or glassy granules. Distorted electrophoresis DNA band patterns will result if the gel is not properly prepared. 11. Electrophoretic separation: The tracking dye should travel at least 6 cm from the wells for adequate separation before staining. 12. Staining: Staining of higher concentration gels (> 0.8%) require additional care to obtain clear, visible results. After staining (15 to 30 min.) with InstaStain Ethidium Bromide or liquid ethidium bromide, examine the results using a UV (300 nm) transilluminator. Repeat the staining as required. Gels stained with InstaStain Methylene Blue or liquid methylene blue stain may fade with time. Re-stain the gel to visualize the DNA bands. 13. DNA 200 bp ladder: After staining the agarose gel, the DNA 200 bp ladder (markers) should be visible. If bands are visible in the markers and control lanes, but bands in the sample lanes are faint or absent, it is possible that DNA was not successfully extracted from the cells. If the ladder, control and DNA bands are all faint or absent, potential problems could include improper gel preparation, absence of buffer in the gel, improper gel staining or a dysfunctional electrophoresis unit or power source.

28 28 Appendix B Polymerase Chain Reaction Using Three Water baths Superior PCR results are obtained using an automated thermal cycler. However, if you do not have a thermal cycler, this experiment can be adapted to use three water baths (Cat. # 544). Much more care needs to be taken when using the three-water bath PCR method. The PCR incubation sample is small and can easily be evaporated. Results using three water baths are often variable. Please refer to the Appendix entitled "PCR Samples with Wax Overlays" for sample handling and preparation tips. Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg +2 and buffer. Preparation of the PCR Reaction: 1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains three critical components: PCR Reaction pellet Primer mix DNA for amplification 2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube. At the start of the PCR reaction, the wax will melt and overlay the samples to prevent evaporation during heating. POLYMERASE chain reaction CYCLING Important Note It is imperative that the temperatures are accurately maintained throughout the experiment. 3. In the three-water bath PCR method, the PCR reaction sample is sequentially cycled between three separate water baths, each set at different temperatures, for a specified period of time. The sequential placement of the reaction sample in the water baths maintained at three different temperatures constitutes one PCR cycle. One example of a PCR cycle might be as follows: 94 C for 1 minute 50 C for 1 minute 72 C for 1 minute See experiment instructions for specific program requirements. 4. The PCR tube must be handled carefully when sequentially cycled between the three water baths. For each cycle: Carefully place the PCR tube in a water bath float. Make sure that the sample volume is at the bottom of the tube and remains undisturbed. If necessary, pulse spin the tube in a balanced microcentrifuge, or shake the tube to get all of the sample to the bottom of the tube. Use forceps to carefully lower the water bath float (with tubes) sequentially into the water baths. 5. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions. On the final cycle the 72 C incubation can be extended to 5 minutes. 6. After all the cycles are completed, the PCR sample is prepared for electrophoresis.

29 Appendix C Preparation and Handling of PCR Samples With Wax For Thermal Cyclers without Heated Lids, or PCR Using Three Water baths 29 Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appropriate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small sample volumes from evaporating. For thermal cyclers without heated lids, or when conducting PCR by the three-water bath method, it is necessary to add a wax bead to the reaction sample. During the PCR process, the wax will melt and overlay the samples to prevent evaporation during heating. Each PCR Reaction pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg +2 and buffer. preparing the PCR Reaction: 1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains the following three critical components: PCR Reaction pellet Primer mix DNA for amplification 2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube. 3. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions. Preparing the PCR Reaction for electrophoresis: 4. After the cycles are completed, transfer the PCR tube to a rack and prepare the PCR sample for electrophoresis. Place the PCR tube in a 94 C water bath long enough to melt the wax overlay. Use a clean pipet to remove most of the melted wax overlay. Allow a thin layer of the wax to solidify. Use a clean pipet tip to gently poke a hole through the solidified wax. Remove the tip. Use another clean pipet tip to enter the hole to remove the volume of mixture specified in the experiment instructions. Transfer this volume to a clean tube. Add other reagents according to experiment instructions, if applicable,. Add 5 µl of 10x Gel Loading solution to the sample and store on ice. 5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as specified in the experiment instructions.

30 30 Appendix D 1.0% Agarose Gel Preparation If preparing the gel with concentrated (50x) buffer, use Table A.1. Table A.1 Individual 1.0% UltraSpec-Agarose Gel Size of Gel (cm) 7 x 7 7 x 14 Amt of Agarose (g) Concentrated Buffer (50X) (ml) Distilled Water (ml) = Total Volume (ml) If preparing the gel with diluted (1x) buffer, use Table A.2. Table A.2 Individual 1.0% UltraSpec-Agarose Gel Size of Gel (cm) 7 x 7 7 x 14 Amt of Agarose (g) Diluted Buffer (1x) (ml) For DNA analysis, the recommended electrophoresis buffer is Tris-acetate-EDTA, ph 7.8. The formula for diluting EDVOTEK (50x) concentrated buffer is one volume of buffer concentrate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit. Table B Electrophoresis (Chamber) Buffer EDVOTEK Total Volume Dilution 50x Conc. Model # Required (ml) Buffer (ml) + M M M36 (blue) M36 (clear) Distilled Water (ml) 294 Table C Volts Time and Voltage (1.0% - 7 x 14 cm gel) Recommended Time Minimum Maximum 55 min 2 hrs 15 min 3 hrs 25 min 1 hr 15 min 3 hrs 5 hrs Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C. The approximate time for electrophoresis will vary from approximately 1-5 hours depending upon various factors. Conduct electrophoresis for the length of time determined by your instructor.

31 Appendix E 1.0% Agarose Gels - Quantity Preparations 31 To save time, electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buffer can be used at a later time and solidified agarose gel can be remelted. Table D Table E Concentrated Buffer (50x) (ml) Amt of Agarose (g) Bulk Preparation of Electrophoresis Buffer + Distilled Water (ml) Total Volume (ml) 60 2, (3 L) Batch Preparation of 1.0% UltraSpec-Agarose Concentrated Distilled + Buffer (50x) + Water = (ml) (ml) Note: The UltraSpec-Agarose kit component is often labeled with the amount it contains. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount. = Total Volume (ml) BULK Electrophoresis Buffer Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D. BATCH Agarose Gels (1.0%) For quantity (batch) preparation of 1.0% agarose gels, see Table E. 1. Use a 500 ml flask to prepare the diluted gel buffer 2. Pour the appropriate amount of UltraSpec-Agarose into the prepared buffer. Swirl to disperse clumps. 3. With a marking pen, indicate the level of solution volume on the outside of the flask. 4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution. 5. Cool the agarose solution to 60 C with swirling to promote even dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step C 6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed. 7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.