PCR Experiment
By Catherine Nikota, G.A. Wheable Secondary School, London, Ontario
Background:
The PCR techniques developed in the mid 1980's are used to amplify a segment of DNA from
a few copies to thousands or millions of copies. To verify that the gene, or segment of DNA,
has been amplified, it is separated into reaction products by agarose gel electrophoresis.
In the DNA lab where the objective of the experiment is insertion of a resistant gene, the
amplified DNA is ligated directly into a standard cloning vector and transformed into bacteria.
Inside the bacteria, the recombinant plasmids are replicated to a high copy number. The
transformed bacteria colonies are isolated and the presence of the insert DNA is verified by
restriction mapping.
Purpose:
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To replicate DNA using a PCR thermal cycler and associated primers
To learn how to load and run a gel electrophoresis
To observe DNA banding produced through the process of electrophoresis and to
compare the PCR sample to the gene ruler provided
Materials:
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PCR thermal cycler
Gel Electrophoresis equipment
UV transilluminator*
Micropipettors and tips
Reaction Mix
PCR Template
Gene Ruler
Ethidium Bromide DNA gel stain
Agarose
TBE buffer
Loading dye
Tube racks
10 ml and 100 ml graduated cylinders
Ziploc bags (medium sized)
Waste bags
* The UV transilluminator will cause intense skin burns and eye damage if turned on without
the shield in place. Ensure that the shield is properly in place before use. Note also that the
glass plate is easily broken. HANDLE WITH CARE.
Biochemistry
PCR Experiment
Procedure:
[There are enough reagents for 10 + 2 experiments] Students can work in groups of up to 3-4
students. Ensure that all reagents are thawed.
Day 1
(allow 20 minutes to prepare samples for PCR machine if each group of students has their own
supply of reaction mix and primer mix)
I. Setting up the PCR reaction
1. Label a 0.2 mL, thin-walled micro tube with the initials of your group and place in ice.
2. Mix reagents gently by flicking with finger.
3. Practice pipetting technique with water and paper towelling, measuring droplet sizes. (Be
sure to depress the pipettor only to the first stop in order to withdraw the precise amount of
mix.)
4. Pipette into the tube:
a. 7.5 ul of PCR reaction mix (includes buffer, dNTPs, enzyme)
b. 7.5 ul of DNA template plus primer mix
5. Keep tube on ice until ready to start the PCR program.
6. Load tubes into PCR machine and start cycling program (cycling parameters have already
been entered into the machine. Program can be left to run overnight.)
7. Freeze the samples after the reaction is finished if samples are not to be used immediately.
Day 2
(allow 1.5 - 2 hours to prepare gel and observe results)
II. Preparation of PCR Reaction for Gel Electrophoresis
1. Pipette 10 ul of the PCR reaction and place in a separate tube.
Biochemistry
PCR Experiment
2. Add 2.5 ul of loading dye (contains a dense chemical called Ficol) to the PCR reaction.
(Low speed gel electrophoresis would provide more explicit results; however, the 30-40
minutes running time can be prohibitive.)
III. High Speed Gel Preparation
1. Mix 10 ml of 10 x TBE with 90 ml of tap water.
2. Pour 80 ml of this solution into the supplied agarose.
3. Swirl to mix.
4. Microwave on high for approximately 30 seconds.
5. Swirl again.
6. Microwave again for another 30 seconds. Watch to ensure that mixture does not boil over!
Solution should be clear and on the point of boiling when the agarose is completely dissolved.
Wearing gloves, continue with next step.
7. Add 8 ul of Ethidium Bromide DNA gel stain (wear gloves as this is a bio hazard!) and allow
gel to cool for a few minutes before pouring the gel in the gel box.
IV. High Speed Gel Box Preparation / Analyzing the PCR Reaction by Gel
Electrophoresis
1. Place metal electrodes in slots provided (Both electrode connectors should be at the top of
the box. The migration will then be from left to right.)
2. Check that the lid will fit later when it is time to run the gel.
3. Place a comb in with the flat side of the comb facing the direction of migration (towards the
positive red electrode). Ensure that the comb is close to, but not touching, the bottom of the gel
box.
4. Pour gel into the box after the solution has cooled for a few minutes.
Biochemistry
PCR Experiment
5. After gel has solidified, pour 40 ml of water over the gel and carefully remove the comb.
6. Set the 2-20 ul pipette to 12 ul and pipette the reaction into a well on a 1% agarose gel.
7. Load 6 ul of the Gene Ruler (DNA) Marker into a separate well.
8. Run the gel at 250 V for 6 minutes (high speed system) or at 150 V for 30-40 minutes (low
speed system)*
* Running a High Speed Gel
1. Ensure connection between electrodes on lid of box and metal posts inside the box are
correct. Be sure buffer or water is in the box. Plug in and turn on power pack. The switch is at
the back.
2. Press the On button on the front display (central button with horizontal line). Adjust voltage
to 240 V and set time to 6 minutes.
3. Press Run.
Note: Once you are finished with the gel and during your wash up procedure, please take care
when washing out the gel box. There is a platinum wire inside the box running along the inside
wall of the box. This wire is delicate and can easily be damaged.
9. Once gel has finished, teacher will remove it from the box and put it into a sealed Ziploc bag
for viewing in the transilluminator.
10. Visualize the Ethidium Bromide DNA gel stain (handle with gloves as this is a bio hazard!),
stained gel under UV light. N.B. Gels are best viewed on the same day they are run. The
bands will diffuse if the gel is left overnight.
* The UV transilluminator will cause intense skin burns and eye damage if turned on without
the shield in place. Ensure that the shield is properly in place before use. Note also that the
glass plate and plexi glass cover are easily broken. Pack and HANDLE WITH CARE.
(Rinse gel apparatus under running tap water.)
Biochemistry
PCR Experiment
Observation Questions
1. There are 3 key temperature changes in a PCR reaction. What are these temperatures?
What is occurring at each of these temperatures?
2. The thermocycler cycles through the 3 temperatures 25-30 times. Why?
3. PCR reactions are performed with Taq DNA polymerase. What is the key feature of this
enzyme that makes it different from most other enzymes?
4. What is the role of the DNA primers in a PCR reaction?
5. It is possible to visualize ~1 ug of DNA on your stained agarose gel. Given that your DNA is
~ 1000 bp, how many molecules is this?
6. In gel electrophoresis, to which pole does the DNA migrate? Why?
7. What is the function of the gel?
8. Why is ice used?
9. Describe 2 practical applications of using the PCR.
Be sure to discuss any experimental errors that may have influenced the procedure.
Biochemistry