1. Gel Electrophoresis Lab
Foodborne Outbreak Investigation

2. Experimental Design
Using your knowledge of PCR and gel electrophoresis, design an experiment to test your hypothesis.
Specifically, how will you tell whether the food item you suspect of being contaminated does indeed contain the microbe?
Keep in mind, there are only 9 wells in the gel. Therefore you have to choose which 9 samples to run.

3. Your Target
You are targeting the ipaH gene and mxiC gene from Shigella sonnei by using the primers specific for these genes in PCR.
When a tiny trace of Shigella sonnei DNA is present in the sample, PCR will yield DNA fragments of 175 bp for the ipaH gene and 1000 bp for the mxiC gene.

4. Available Supplies

5. Reference DNA
The SRS, M1M, & 1 kb Ladder samples are provided because they produce fragments of known size that can be compared to the PCR products from the five layer dip samples.
You do not need to use all three of these reference DNA samples.

6. Remember to use controls!
The negative control includes no DNA template and therefore should not yield visible bands.
Because certain molecules in food can inhibit PCR causing it to fail (especially when trying to detect a low number of bacteria in the sample), a positive control is included within each bean dip layer and the mixed sample to differentiate between a negative result due to lack of Shigella in the sample and a negative result due to total failure of the PCR reaction.
The positive control is a lab-created strain of Shigella not found in nature. This strain also produces the 175 bp fragment for the ipaH gene, but the mxiC gene has been modified to produce a fragment of 1800 bp.

7. Running the Gel
The phosphate groups in the sugar-phosphate backbone of DNA are negatively-charged.
Molecules move through the gel at different rates, determined largely by their size and charge. Shorter molecules move through the gel faster than longer molecules.

8. Hypothetical Gel
+1C
SRS
M1M
2000
1000
500
300
100

9. Loading the Gel
Follow the “How to Use a Micropipette” instructions at your station.
When finished, eject the tip into the “Trash” disposal at your station.
Carefully handle the DNA samples!
Be sure your gel is fully submerged in the buffer solution.
Hold the micropipette just over the
well. Be careful not to puncture
the bottom of the well with the
micropipette!