Water pollution is a universal problem because clean water is essential for human health, aquatic life, and agriculture. Although drinking water in U.S. cities is generally safe, monitoring remains necessary because our water supply can still be contaminated by corrosion in aging water pipes and other environmental factors. One environmental threat to the water system is represented by bacteria and other microorganisms. These microbes, when ingested, can cause severe illness. For example, in 1993 the protozoa Cryptosporidium parvum caused gastrointestinal distress in over 400,000 individuals in Milwaukee, Wisconsin. The outbreak resulted in 4,000 hospitalizations and more than 50 deaths. Another example is Escherichia coli. Most strains of E. coli are relatively harmless or aid digestion in human beings and other animals. However, certain strains of the bacterium (O157:H7 in particular) produce a potent toxin that can cause acute bloody diarrhea, kidney failure, and even death.
We can test for these microbes using the Polymerase Chain Reaction (PCR), a biotechnology technique that allows researchers to quickly create billions of copies of a specific region of DNA in vitro. These tests can even be performed in the classroom laboratory using Edvotek Experiment #953, Multiplex PCR Analysis for Water Contaminants. This popular experiment is used by educators around the world to talk about water contamination. In this post, we’ll talk about culturing bacteria, splitting the kit for multiple sections, and best stopping points for performing the kit over several days.
Module I: Isolation of Bacterial DNA from Contaminated Water
- Three different bacteria are included with this experiment in BactoBead form – B. subtilis, E. coli, and S. marcescens. The BactoBeads are mixed with water and incubated for 15 minutes before class. If you need to perform the experiment over multiple days, the cultures can be stored in the refrigerator for up to one week. Before aliquoting, gently shake the cultures to resuspend the bacteria.
- The lysis buffer is prepared before class by mixing the Proteinase K with the Universal DNA buffer (component A). After preparation, the buffer should be aliquoted and stored on ice before use. If you have sections on multiple days, freeze the buffer in individual aliquots until needed.
- If you are using environmental samples, they should be collected as close to the class as possible. Please note, while the experiment tests for bacteria commonly found in water (like E. coli), samples collected from local water sources may not contain any of the bacteria identified by this experiment.
- If you do not have time to proceed to Module II, the samples can be stored at -20ºC until the next class.
Module II: Multiplex PCR Amplification of Water Contaminants
- Before class, the primers and control DNA can be diluted, aliquoted, and stored on ice. If you have sections on multiple days, freeze the individual aliquots at -20ºC. Diluted primers can degrade over time, so we do recommend using them in PCR within a week of preparation for best results.
- If you do not have time to start thermal cycling, do NOT add the PCR EdvoBead! The freeze-thaw can ruin the Taq polymerase, which is the enzyme that builds DNA. This will affect the results of your PCR experiment. Instead, freeze the primer-DNA mix overnight and add the PCR bead right before amplification.
- After the amplification, the DNA is stable. If you run the thermal cycler overnight, end your PCR program with a static temperature hold from 4ºC to 16ºC. Once removed from the thermal cycler, the samples can be frozen until you are ready to perform electrophoresis.
Module III: Separation of PCR Products by Electrophoresis
- Electrophoresis is a very forgiving technique in terms of preparing in advance. The gels can be prepared in advance and stored in the refrigerator in buffer to prevent them from dehydrating. Place the gels in plastic bags and add 2 mL of buffer – keep the buffer volume low so that the SybrSafe does not diffuse out of the gel.
- The diluted buffer can be stored at room temperature until needed.
- After performing electrophoresis, SybrSafe gels can be visualized immediately. If you want to look at the gels later, they can be placed back in the plastic bag with a small amount of buffer and stored in the refrigerator for a few days.
- If you are staining gels with FlashBlue, the gels can be stained overnight in a dilute solution of the DNA stain and visualized the next day. If you want to look at the gels later, they can be placed back in the plastic bag with a small amount of buffer and stored in the refrigerator for a few days. If the bands fade, the gels can be restained.
If you have any more questions about the protocol, email us at firstname.lastname@example.org or call our technical support team at 1.800.338.6835.