Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

1. Sample Collection

1. Collect soil using an auger or shovel down to a determined depth. If collecting soil from the rhizosphere (the narrow region of soil surrounding and influenced by plant roots and their associated microbes), only collect directly from around the plant roots by hitting the soil off into a collection barrel.
2. Collect water sample by dipping a sterile plastic bottle into the water while holding the end of the dipping stick.

2. Nucleic Acids Extraction and Preparation

1. Collect organisms and viruses from the sample, and extract DNA and RNA from them. For details, please refer to the JoVE Science Education video on community nucleic acid extraction.
2. Store the extracted DNA in labeled microfuge tubes. If the DNA needs to be stored overnight or for longer periods of time, freeze it at -20 °C, and thaw the tubes at room temperature when ready for use.
3. If the genetic material to be assayed is RNA (whether it is the genome of RNA viruses, or the transcribed RNA of cellular organisms), perform reverse transcription (RT) on the sample to create complementary DNA (cDNA) before proceeding to PCR. For details, please refer to the JoVE Science Education video on RT-PCR.

3. Polymerase Chain Reaction

1. Place the PCR enzyme (e.g., Taq polymerase) on ice and thaw the other reagents (PCR buffer, dNTPs, primers) inside a designated “clean” hood at room temperature. The enzyme is stored at -20 °C but never freezes. It is temperature sensitive, and so must be kept cool and its exposure to ambient temperature minimized
2. Calculate the volume of each reagent needed to make a “master mix” of all reagents that are constant among all reactions (Table 1). Make sure to account for positive (e.g., a template known to contain the target region) and negative (e.g., no template) controls in the calculations. Add an additional 10% to the final volume to account for pipetting error. Primer volumes depend on assays for specific organisms; refer to published literature for the appropriate values. 
3. Using a low-binding microfuge tube, which minimizes the adhesion of reagent molecules to the plastic walls, add the calculated volumes of each reagent to assemble the master mix. Gently vortex and centrifuge each reagent before adding. Once the master mix is prepared, vortex to mix and collect by centrifugation
4. Prepare 8-tube PCR strips. Designate a tube for each sample, including positive and negative controls
5. Dispense the appropriate volume of PCR mixture into each tube of the strip
6. Add the appropriate volume of DNA template from the samples, as well as 5 μL of the positive template and 5 μL of molecular grade water as negative control, into the respective PCR tubes.
7. Place the cap securely on the 8-tube strip and centrifuge for a few seconds using a minicentrifuge
8. Place the 8-tube strip in a thermocycler
9. Set the appropriate PCR program to run on the thermocycler. A typical program consists of the following
   1. Denaturation at 94 °C for 3 min.
   2. 30–40 cycles of amplification: denaturation at 94 °C for 30 s, annealing at primer-specific temperature (usually between 50–60 °C) for 30 s, and extension at 72 °C for 30 s / 500 bp.
   3. Final extension at 72 °C for 7-10 min. 

4. Agarose Gel Preparation

1. Based on the desired gel volume and gel concentration (Table 2), weigh out the appropriate amount of agarose powder into a 125-mL Erlenmeyer flask.
2. Add the appropriate volume of gel-running buffer into the flask, and swirl the flask by hand.
3. Heat the buffer-agarose mixture in a microwave oven at high power for 1 min.
4. Remove flask from the microwave and swirl by hand to make sure all the agarose has dissolved. If the agarose has not completely dissolved, repeat microwaving in 30-s increments.
5. After tightly securing the cap onto the flask, cool it to 50 °C by rotating under running cold water.
6. Add 1 μL of ethidium bromide (EtBr) to the agarose mixture using a micropippette designated for EtBr. EtBr is a dye that binds double-stranded nucleic acids and fluoresces orange when illuminated with UV light. Note that EtBr is potentially carcinogenic, so personal protective equipment (goggles, lab coat, EtBr resistant gloves) must be worn.
7. Pour the molten gel into an electrophoresis gel casting tray. Make sure that no bubbles are trapped within the agarose. Place a comb into the gel and clamp securely. Wait approximately 20-30 min for the gel to solidify.
8. Once the gel has solidified, remove the comb carefully without causing any tears in the gel. The comb creates wells in the gel for loading the samples.

5. Gel Electrophoresis

1. Place the solidified agarose gel into the electrophoresis chamber.
2. Add the appropriate running buffer into the chamber until the gel is barely submerged.
3. Onto a piece of Parafilm, pipette spots of loading dye concentrate. Also include a DNA ladder of a suitable range for the expected sizes of the PCR products. Alternatively, use a fresh set of microfuge tubes, one for each sample.
4. Once the PCR is completed, retrieve that 8-tube strip from the thermocycler, and briefly centrifuge to collect any condensates. Add an appropriate volume of DNA product to the loading dye and pipette up and down to mix. For example, 2 µL of 10x loading dye is mixed with 8 µL of sample to give a final volume of 10 µL, with the dye at 1x.
5. Pipette each dye-sample mixture into the designated wells in the agarose gel, being careful not to puncture the wells.
6. Connect the electrodes to the electrophoresis chamber. DNA is negatively charged, so it “runs” toward the positive electrode. Therefore, connect the positive electrode to the opposite side of the chamber, where the wells were loaded. Set the power supply to a voltage appropriate for the buffer system and size of the gel chamber, and set it to run. Small bubbles should be visible moving up the sides of the chamber if the electrophoresis is proceeding properly.
7. Once the dye front has advanced far enough down the gel, turn off the power supply. Carefully transport the gel into the transilluminator or visual imager and turn on the UV light to visualize the DNA bands on the gel.
8. Analyze the band size and positions on the gel. Compare the band positions of the samples to the positive control to determine if DNA from the organism of interest is present in the sample (Figure 1).

Component	Volume per Tube (μL)	Volume for 5 tubes (μL)	Final Concentration
10x Ex Taq buffer	5.0	25	1x
2.5 mM dNTPs	4.0	20	0.2 mM
Forward Primer*	2.0	10	400 nM
Reverse Primer*	2.0	10	400 nM
Molecular H2O	31.75	158.75	-
Ex Taq	0.25	1.25	2.5 U
PCR Mixture	45	225

Table 1. Reagent volumes for PCR master mix. *Primer volumes vary depending on organism assay. Adjust the volume of molecular grade water to make the final volume 45 μL. Volumes of other components should not vary.

Recommended % of Agarose	Optimum Resolution for Linear DNA Fragments (base pairs)
0.5	1,000-30,000
0.7	800-12,000
1.0	500-10,000
1.2	400-7,000
1.5	200-3,000
2.0	50-2,00

Table 2. DNA fragment size ranges optimally resolved by different agarose gel percentages.