INTRODUCTION TO PCR AND ELECTROPHORESIS
Purpose:
The purpose of this laboratory is to amplify and characterize student DNA samples using
polymerase chain reaction (PCR) methods and gel electrophoresis.
Discussion:
Answer the following questions to develop your procedure:
1. What is PCR? Who developed the process? What makes the process better than
previous techniques?
2. What are some common uses of PCR?
3. How does PCR work to amplify a DNA strand?
4. What are the general procedures for PCR?
5. What portion of DNA does this lab test for? How are the results analyzed to
determine whether or not this portion of DNA is present?
6. What is electrophoresis? How does it work? How is it used in this lab?
Objectives:
In this lab, you will…
1. amplify cheek cell DNA using polymerase chain reaction (PCR),
2. run the amplified cheek cell DNA through gel electrophoresis,
3. and determine if students are homozygous (+/+), homozygous (–/–), or
heterozygous (+/–) for a single Alu sequence within the PV92 locus on
chromosome 16.
Safety:
Most (if not all) of the equipment used in this lab is borrowed from Clarion University, so
please handle with care. The equipment is borrowed because we cannot afford it, therefore we
cannot afford to replace it.
Procedure:
Part 1: Cheek Cell DNA Template Preparation
Materials:
Equipment:
Screwcap tubes
Microcentrifuge tubes
Micropipets w/ filter tips
Digital dry bath
Cups
Fine-tip marker
Chemicals:
InstaGene matrix
1. Label one screwcap tube with your initials. Pipet 200 L of the InstaGene matrix into the
screwcap tube.
2. Rinse your mouth thoroughly with tap water.
3. Scrape the inside of your cheek with a sterile micropipet tip to get a “plug” of cheek cells.
4. Put the micropipet tip on a micropipet and insert the tip containing the “plug” of cells into
the InstaGene matrix in your screwcap tube. Push the pipet up and down to get the cells
to fall into the InstaGene matrix.
5. Spin your tube in a balanced centrifuge at full speed for 2 minutes. When the centrifuge
has completely stopped, remove your tube. You should see a match-head sized pellet of
whitish cells at the bottom of the tube. If you don’t see a pellet of this size, scrape more
cells from your cheek using the micropipet tip, mix the cells into the InstaGene matrix,
and repeat the spin.
6. Resuspend the pellet by flicking the tube so that no clumps of cells remain.
7. Screw the cap tightly on the tube. Shake or vortex to mix the tube contents.
8. When all members of the class are ready, place the tubes in the digital dry bath, and
incubate at 56°C for 10 minutes. At the halfway point, shake or vortex the tubes gently,
then place them back in the dry bath for the remainder of the time.
9. Remove the tubes, and set the digital dry bath to 100°C. Shake or vortex the contents of
the tube. Incubate at 100°C for 5 minutes.
10. Remove the tubes from the boiling water bath and shake or vortex the contents to
resuspend. Pellet the matrix by spinning at 6 rpm for 5 minutes in a centrifuge.
11. Store your screwcap tube in the refrigerator for Part 2.
Part 2: PCR Amplification
Materials:
Equipment:
Microcentrifuge tubes
Screwcap tubes
12 PCR tubes
Micropipets w/ tips
Fine tip marker
Microcentrifuge
Thermal cycler
Chemicals:
Master mix
Primer mix
PV92 homozygous (+/+) control
PV92 homozygous (–/–) control
PV92 heterozygous (+/–) control
12. Label a capped micro test tube “MM” for master mix. Dispense 550 L of the master
mix into the capped micro test tube. Pipet 11 L of the primer mix to the master mix and
vortex for 10 seconds to mix. This will be the master mix to be used for the entire class.
13. Label three microcentrifuge tubes as +/+, +/–, and –/–. Pipet 20 L of each control into
these labeled tubes. Pipet 20 L of the master mix into these tubes as well.
14. Obtain the tube with your DNA template from the refrigerator. Spin the screwcap tube
for 2 minutes at 6 rpm in the centrifuge.
15. Label a microcentrifuge tube and a capless micro test tube with your initials, place the
PCR tube in the capless tube, and place both in the foam holder.
16. Pipet 20 L of your DNA template (the supernatant) from the screwcap tube into the
bottom of the PCR tube. Be very careful not to transfer any of the matrix beads into the
PCR tube.
17. Transfer 20 L of the master mix into the PCR tube. Mix by pipetting up and down 2-3
times. Cap the PCR tube tightly. The mixture should be yellow.
18. Program the thermal cycler so that it runs the following cycles:
Cycle 1:
Step 1: 94°C for 1 minute.
Run only once.
Cycle 2:
Step 1: 94°C for 1 minute.
Step 2: 60°C for 1 minute.
Step 3: 72°C for 2 minutes.
Repeat 40 times.
Cycle 3:
Step 1: 72°C for 10 minutes.
Run only once.
19. Place the PCR tube into the thermal cycler along with the +/+, +/–, and –/– controls.
20. Place the PCR tubes in the refrigerator after thermal cycling.
Part 3: Electrophoresis of Amplified PCR Samples and Staining of Agarose Gels
Materials:
Equipment:
Microcentrifuge
Micropipets w/ filter tips
Electrophoresis equipment:
Gel boxes
Casting trays
Combs
Wires
Power supply
Staining tray
Chemicals:
Orange G loading dye
Agarose gel
Electrophoresis buffer (50x TAE)
Master mix
Primer mix
PV92 homozygous (+/+) control
PV92 homozygous (–/–) control
PV92 heterozygous (+/–) control
21. Prepare 1 L of 1x TAE buffer by adding 20 mL of 50x TAE buffer to a 1 L volumetric
flask and dilute to the mark with distilled water. Make 2 L of buffer.
22. Prepare the agarose solution by adding 3.5 g of agarose to a 1000 mL Erlenmeyer flask,
then pouring 350 mL of 1x TAE buffer into the flask. Swirl to suspend the agarose, then
heat it on a hot plate to boiling. Let cool to 60°C before pouring the gels.
23. Tape the end of the gel casting tray to create a fluid-tight seal. Insert the gel combs so
they are ~ 2 cm from the end of the casting tray. Allow the gel to solidify at room
temperature for 10-20 minutes. Carefully remove the comb from the solid gel, and
carefully remove the tape from the edges of the tray.
24. Obtain your PCR tube and the controls from the refrigerator. Pulse-spin the tube for ~3
seconds at 2 rpm in the centrifuge.
25. Add 10 L of Orange G loading dye into your PCR tube and the control mixtures, and
mix gently.
26. Place an agarose gel in the electrophoresis apparatus. Check that the wells of the agarose
gels are near the black (–) electrode and the base of the gel is near the red (+) electrode.
27. Using a clean tip for each sample, load the samples into 8 wells of the gel in the
following order:
Lane 1: MMR (DNA standard)
10 L
Lane 2: Homozygous (+/+) control 10 L
Lane 3: Homozygous (–/–) control 10 L
Lane 4: Heterozygous (+/–) control 10 L
Lane 5: Student 1
20 L
Lane 6: Student 2
20 L
Lane 7: Student 3
20 L
Lane 8: Student 4
20 L
28. Fill the electrophoresis chamber and cover the gel with 1x TAE buffer. Pour very slowly
as to avoid disturbing the samples in the wells.
29. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red
to red and black to black. Connect the electrical leads to the power supply.
30. Turn on the power supply and electrophorese your samples at 100 V for 30 minutes.
31. When electrophoresis is complete, turn off the power and remove the lid from the gel
box. Carefully remove the gel tray and the gel from the gel box. Be careful, the gel is
very slippery. Nudge the gel off the gel tray with your thumb and carefully slide it into
your plastic staining tray.
32. Add enough 50x Fast Blast DNA stain to your staining tray to submerge your gel. Leave
the stain in the tray for 15 minutes.
33. The stain can be recycled multiple times. Pour the stain back into the original container.
34. Rinse the stain three times with warm tap water. Add enough warm tap water to
submerge your gel. Let stand for 15 minutes. Repeat this process until the bands on the
gel are clear enough to analyze.
35. Place the gel on a light box and record your result. Photographing or sketching the gel is
adequate.
Cleanup:
Analysis:
1. Use your gel to determine if you are homozygous (+/+), homozygous (–/–), or
heterozygous (+/–) for the Alu insert.
 The EZ Load DNA MMR ruler contains 1000 bp, 700 bp, 500 bp, 200 bp and 100
bp fragments.
o If you are homozygous (+/+), you will have a single band at ~941 bp.
This means you have the Alu insert on both chromosomes.
o If you are homozygous (–/–), you will have a single band at ~641 bp. This
means you do not have the Alu insert on either chromosome.
o If you hare heterozygous (+/–), you will have a band at ~941 bp and a
band at ~641 bp. This means you have the Alu insert on one chromosome,
but not the other.