Mitochondrial DNA extraction from cheek cells & HotStart PCR
This lab session has 3 parts:
1. Extract DNA from student cheek cells and set up PCR for mtDNA HVR1
2. Wet mount cheek cells on slide, stain and observe under microscope
3. Cast 1.5% agarose gels in TBE containing GelRed
Materials to be prepared:
Each bench should have an ice bucket with:
 10% Chelex solution (Carolina) OR Instagene Matrix (Biorad), 0.4 mL aliquot in 2.0mL screwcap tube (be sure to pipet immediately after vortexing, to get even delivery of
Chelex beads into every tube.
 Q-tips or cotton-tipped applicators, 1 per student
 0.5 mL saline solution (0.9%) in 1.5-mL microcentrifuge tube, 1 per student
 HotStart PCR pre-mix tube, 1 per student
Other items:
 sterile 1.5 mL microcentrifuge tubes, sterile pipet tips, set of pipetters
 glass slides and cover slips - 1 per student
Set up boiling water baths: 1-L beakers with floats for 1.5-mL tubes
Thaw human mtDNA primer mix (Carolina Biologicals cat# 211504) and set in TA's ice bucket
Procedure overview:
DNA extraction from cheek cells - per Q-tip protocol
PCR - each student adds 18 microliters human mtDNA primer mix to HotStart PCR tube
Microscopy of cheek cells
Q-Tip DNA Extraction from Cheek Cells
Each person does the following:
1. Label all your plastic microfuge tubes with your assigned ID number on both side and top.
2. Label plastic microfuge tube containing clear saline solution "CC" and your ID number.
3. Swab inside of your cheek vigorously with Q-tip to collect cheek cells - 5 sec.
4. Twirl end of Q-tip with cheek cells in the "CC" tube's saline solution, back and forth,15 sec.
5. Remove Q-tip and inspect saline solution. Cheek cells should create turbidity (cloudiness).
6. Transfer 200 microliters of cheek cell suspension to screwcap tube containing 0.4 mL Chelex
bead mixture. Be sure this screwcap tube has your ID number on the top!
7. Save and set aside the remaining cheek cell suspension in the CC tube on ice for microscopy.
8. Screw cap on Chelex tube firmly, and vortex vigorously to mix cells with Chelex beads.
9. Put screwcap tube with cheek cells and Chelex in boiling water bath 5 min.
10. Cool on ice or on benchtop, 2 min.
11. Centrifuge full-speed 1 min.
12. Without disturbing pellet at bottom of tube, transfer 0.1 mL (100 microliters) of clear
supernatant to new 1.5-mL tube (label "DNA" with your assigned ID number). This is your
crude cheek cell DNA sample.
PCR Amplification of human mitochondrial DNA (mtDNA)
1. Use a micropipet with a fresh tip to add 18.0 µL of mtDNA primer/loading dye buffer mix to a
HotStart pre-mix PCR reaction tube.
2. Use a fresh tip to add 2.0 µL of your DNA sample to the PCR reaction tube, and pipet up and
down or flick it to mix. Pool reagents by pulsing in a microcentrifuge or by sharply tapping the
tube bottom on the lab bench.
3. Label the side and cap of your PCR tube with your assigned ID number.
4. Load PCR tube into the thermal cycler.
5. Program and start thermal cycler with a step file:
94°C 30 sec
58°C 30 sec
72°C 50 sec
30 cycles
72°C 5 min extension
4°C 99 min x 99 cycles (overnight soak)
Wet mount and staining of cheek cells
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Add one drop of methylene blue dye solution to your cheek cell suspension (CC tube)
and resuspend cells.
Add one drop (or 50 µL) of cell suspension with dye on a clean glass slide.
Put coverslip over drop of cells, starting from one edge, and wick off excess moisture.
Observe under microscope at 10X and 40X.
Microscopy
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Start with 10X objective.
Place slide on stage & scroll horizontally and vertically to place one edge of the
coverslip under the objective.
Lower objective until it is just above the coverslip.
While viewing through the eyepiece, slowly raise the objective until the field of view
comes into focus - the edge of the coverslip should be in sharp focus.
Scroll the slide across the stage to view the stained cheek cells. You should be able to
see squamous epithelial cells, with dark round spots in their centers, corresponding to
the nucleus of the cell..
Switch to 40X objective to increase magnification. These scopes are par-focal, meaning
that you should not have to adjust the focus much, if at all, when you switch objectives.
Observe darkly stained nuclei in the squamous epithelial cells.