Module Mystery 3 – The Case of the Bad Burger
The Case: Just when the poor folks in Virulent, Pennsylvania thought it was safe to go
back to their daily lives, disease struck again! Approximately fifty people fell ill during a
forty-eight hour period. Their symptoms included severe abdominal pain, nausea,
cramping and vomiting. Health officials quickly ascertained that all fifty affected people
had recently eaten at "Buff's Big Bad Burgers", where they had each consumed a double
bad-boy burger. Buff loudly denied that his burgers could be to blame, and has agreed to
let you test the burgers for the presence of E. coli DNA.
Your Job: The Virulent Department of Health needs to know if the hamburger is indeed
the source of the food poisoning, and, if so, which bacterial species is responsible. This
information can then be used to determine the probable source of the contamination. The
following is a list of bacteria that are considered likely “suspects”:
Salmonella enterica (serotype typhimurium)
Campylobacter jejuni
Escherichia coli
Listeria monocytogenes
During the next three lab periods, you will 1) determine if the hamburger was
contaminated using the polymerase chain reaction, 2) determine the type of bacterial
contamination and, 3) support your conclusions using BLAST analysis of your gene
sequences.
Lab Report: The oral presentations for this module mystery will be during Week 14
(December 1-5).
Procedure:
Week One: Polymerase Chain Reaction
In a 0.2 mL microcentrifuge tube, add:
25 L Ready Mix PCR Reaction Mix
2 L forward primer
2 L reverse primer
1 L hamburger DNA
20 L sterile water
In a separate tube, add:
Same as tube one, but 1 L of sterile water
in place of the hamburger DNA (yes, this is
your negative control).
Place tubes in the thermal cycler and start FILE  CK
Conditions of file:
95°C for 4 min., then
94°C 1 min
55°C 1 min.
Repeat 30 cycles
72°C 2 min
then 72°C for 5 min.
Week Two:
Analysis of Products
1) Run a 1% agarose gel with EtBr in 1 X TAE to determine the size of your fragment(s).
You will use the Hin dIII-digested Lambda DNA standard.
2) Load 5 uL of each sample, including the size standard, into each well and run at
approximately 100 volts until the dye has migrated about half way down the gel.
3) Take a picture of your gel. Is the bacterial 16S rRNA gene fragment (around 798 bp)
visible in any of your samples?