Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
miniPCRTM Crime Lab: Missy Baker Gone Missing
Missy Baker has gone missing. Two suspects are held by the police while hair samples found in
their cars is analyzed by PCR to evaluate whether their DNA matches the victim’s DNA.
1|miniPCRTM Crime Lab
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
Experimental instructions
A. PCR set up
1. Label 4 PCR tubes (200 µL) per lab group

1 tube labeled “A”: ‘Hair DNA’ extracted from Suspect A’s car
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1 tube labeled “B”: ‘Hair DNA’ extracted from Suspect B’s car
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1 tube labeled “H”: ‘Control DNA’ from a healthy individual
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1 tube labeled “D”: 'Control DNA' from a CFTR deletion mutant

Each tube should also be labeled with the group’s name
Alternatively, if your class is divided in 2 types of groups, label 2 plastic tubes


“Detectives” groups

1 tube labeled “A”: ‘Hair DNA’ extracted from Suspect A’s car

1 tube labeled “B”: ‘Hair DNA’ extracted from Suspect B’s car
“Reference Labs” groups

1 tube labeled “H”: ‘Control DNA’ from a healthy individual

1 tube labeled “D”: 'Control DNA' from a CFTR deletion mutant
2. Add the following to each 200 µL tube

PCR Mix
15 µL per tube
(Same for all reaction tubes)

DNA Primer Mix
10 µL per tube
(Same for all reaction tubes)

Template DNA sample
5 µL per tube
(A, B, H, or D depending on the tube)
3. Cap and gently mix or flick the reaction tubes

Make sure all the liquid volume collects at the bottom of the tube by
gently flicking or by briefly spinning the tubes using a microcentrifuge
4. Place the tubes inside the PCR machine

Close the lid and gently tighten it
2|miniPCRTM Crime Lab
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
B. PCR programming and monitoring (illustrated using miniPCRTM software)
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1. Open the miniPCR software app and remain on the "Protocol Library" tab
2. Click on the "New Protocol" button on the lower left corner

Optional: select existing “miniPCR Forensics Lab” protocol, skip to step 7
3. Select the PCR "Protocol Type" from the top drop-down menu
4. Enter a name for the Protocol; for example "Group 1 - Crime Lab"
5. Enter the PCR protocol parameters:
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Initial Denaturation
Denaturation
Annealing
Extension
Final Extension
Number of Cycles
Heated Lid
3|miniPCRTM Crime Lab
94°C, 30 sec
94°C, 5 sec
57°C, 5 sec
72°C, 10 sec
72°C, 30 sec
30
ON
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
6. Click "Save" to store the protocol
7. Click “Upload to miniPCR” (and select the name of your miniPCR machine in the
dialogue window) to finish programming the thermal cycler. Make sure that the
power switch is in the ON position.
8. Click on “miniPCR [machine name]” tab to begin monitoring the PCR reaction
9. miniPCRTM software allows each lab group to monitor the reaction parameters in
real time, and to export the reaction data for analysis as a spreadsheet.
10. At the end of the run, the screen will show “Status: Completed” and all LEDs on
your miniPCR machine will light up.
 You can now open the lid and remove your tubes, being careful not to
touch the metal lid which may still be hot
4|miniPCRTM Crime Lab
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
C. Gel electrophoresis – Pouring agarose gels (Preparatory activity)
Note: If the lab is going to be completed in one lab session, agarose gels should be
prepared immediately after PCR is started to allow the gel to set.
If the lab is going to be performed over two classes, gels can be prepared up to one day
ahead of the second class and stored in a refrigerator, covered in plastic wrap.
A. Prepare a clean and dry agarose gel casting tray

Seal off the ends of the tray as indicated for your apparatus

Place a well-forming comb at the top of the gel (10 lanes or more)
B. For each experimental group, prepare a 1.6% agarose gel using 1X TAE buffer

Adjust volumes and weights according to the size of your gel tray

For example, add 0.8 g of agarose to 50 ml of 1x TAE buffer

Mix reagents in glass flask or beaker and swirl to mix
C. Heat the mixture using a microwave or hot plate

Until agarose powder is dissolved and the solution becomes clear

Use caution, as the mix tends to bubble over the top and is very hot
D. Cool the agarose solution for about 2-3 min, swirling intermittently
E. Add gel staining dye
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Follow dye manufacturer instructions
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Typically, 1.0-10.0 µL of staining dye per 100 mL of agarose solution

Note: Follow manufacturer’s recommendations and state guidelines if
handling and disposing of ethidium bromide
F. Pour the cooled agarose solution into the gel-casting tray
G. Allow gel to completely solidify (until firm to the touch) and remove the comb

Typically, 15 minutes
H. Place the gel into the electrophoresis chamber and cover it with 1X TAE buffer
5|miniPCRTM Crime Lab
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
Gel electrophoresis – Running the gel
1. Make sure the gel is completely submerged in TAE

Ensure that there are no air bubbles in the wells (shake the gel
gently if bubbles need to be dislodged)

Fill all reservoirs of the electrophoresis chamber and add just
enough buffer to cover the gel and wells.
2. Add 6 µL of 6X gel loading buffer into each PCR tube and DNA ladder
3. Load DNA samples onto the gel in the following sequence

Lane 1: 10 µL DNA ladder
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Lane 2: 15 µL PCR from Suspect A DNA
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Lane 3: 15 µL PCR from Suspect B DNA
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Lane 4: 15 µL PCR from Control H DNA
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Lane 5: 15 µL PCR from Control D DNA
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Lane 6: 10 µL DNA ladder
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Note: you can load PCR samples from other groups before the ladder
4. Place the cover on the gel electrophoresis box

Ensure the electrode terminals fit snugly into place
5. Insert the black and red leads into the power supply
6. Set the voltage at 100-130V and conduct electrophoresis for 15 minutes, or until
the loading buffer dye has progressed to at least half the length of the gel

Check that small bubbles are forming near the terminals in the box

Longer electrophoresis times will result in better size resolution

Lower voltages will result in longer electrophoresis times
7. Once electrophoresis is completed, turn the power off and remove the gel from
the box
6|miniPCRTM Crime Lab
Version: 2.4
Release: January 2015
© 2013-2015 by Amplyus LLC
D. Size determination and interpretation
1. Place the gel on the transilluminator

If using UV light, cover any exposed skin and cover eyes with UVprotective goggles
2. Verify the presence of PCR product
3. Ensure there is sufficient DNA band resolution in the 400-800 bp range of the
DNA ladder

Run the gel longer if needed to increase resolution
4. Document the size of the PCR amplified DNA fragments

Capture an image with a smartphone camera

If available, use a Gel Documentation system
7|miniPCRTM Crime Lab