PCR Forensics. Today’s Lab There has been an outbreak of Salmonella poisoning in the Student Union cafeteria at Stanford University cafeteria. You have.

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Presentation transcript:

PCR Forensics

Today’s Lab There has been an outbreak of Salmonella poisoning in the Student Union cafeteria at Stanford University cafeteria. You have been asked to identify the source of the Salmonella. Possible contaminated foods are: bad salami, unwashed lettuce, contaminated hamburger meat. You extract DNA from the three potentially contaminated foods. You also have a positive control (DNA from sample known to contain salmonella) and a negative control (DNA from sample known to not contain salmonella). Your job is to figure out which foods are contaminated. You will use PCR to figure out which sample is contaminated.

The 3 Steps of PCR 1. Melt: Heating double-stranded DNA causes the strands to separate. (95C) 2. Anneal: Cool to bind the primers to single stranded DNA. (65C) 3. Extend: Let polymerase extend off the primer (72C) 4. Back to step 1. Repeat 20 to 40 times. (See video)

Ingredients DNA template - DNA you want to amplify from Primers - bind to DNA and replication begins at these primed regions. Two primers are created specifically for the area of interest. Buffer - provides salt and pH Deoxynucleotide-triphosphates (dNTPs) - Mixture of dATP, dGTP, dTTP, dCTP, building blocks of DNA Mg 2+ - cofactor for Taq, also changes salt conditions for reaction. Taq Polymerase

How do we do so many cycles? 95 degree - melt 65 degrees - anneal primers 72 degree extension x Now we use a machine called the thermocycler

Use Gel Electrophoresis to observe different fragment lengths. 1.Load fragmented DNA into well. 2.Run a current through the gel. Negative at top, positive at bottom. DNA is negatively charged. 3.Large fragments move slowly, small fragments move fast. 4.Stain DNA to visualize.

Lab Setup Use 5 thin-walled PCR tubes To each tube, add 10uL DNA from one food source, positive control, or negative control Add 15uL primer mix (forward and reverse primer already mixed for you) Add 25uL PCR master mix (contains buffer, Mg++, dNTPs, Taq polymerase)

Running PCR reaction Put your tubes in the PCR thermocycler and run with these conditions: 94 o C 2 minutes (to melt DNA) 94 o C 30 seconds 50 o C 30 seconds x35 cycles to amplify 72 o C 30 seconds 72 o C 7 minutes 4 o C hold

Running a Gel

Expected results Salmonella genomic DNA in food sample Primer 1 Primer 2 Amplified Salmonella DNA (511 bp)

Expected results Positive control: band Negative control: no band Food samples: A DNA band indicates presence of Salmonella DNA.