Polymerase Chain Reaction (PCR) and Gel
Electrophoresis
EXPERIMENT 3
In this experiment you will:
(a) use PCR to amplify a plasmid form of DNA called pCDNA3.1(+)/GFP Plasmid
DNA.
(b) run the DNA products formed during PCR on an agarose gel.
Materials
PCR:
Target DNA: pCDNA3.1(+)/GFP Plasmid
DNA, 985 ng/l
dNTPs (deoxynucleotide phosphates), 10
mM
Up primer, 1.22 nmol/l
Low primer, 1.54 nmol/l
Taq DNA polymerase, 5000 units/ml
Buffer A, 10X
Molecular biology grade water (milliQ
water)
Styrofoam cooler
Crushed ice
0.2 ml PCR tubes
Eppendorf tubes
Micropipettors and sterile tips
Microcentrifuge
Thermocycler
Gel Electrophoresis:
Agarose
TAE (Tris-acetate EDTA) buffer, pH 8.0, 1X
Loading dye (blue juice)
Syber® Safe, 10,000X
Molecular weight markers (DNA ladder)
Microwave
Balance, weigh boats, spatulas
Graduated cylinder, 500 ml
250 ml media bottle and cap
Hot hands protector
Electrophoresis box, power supply,
comb, dams
Parafilm, 3 small squares
Gel photo imaging system
Procedure
Part A. Get Ready
1. As specified in the Laboratory Notebook Preparation handout, setup your lab
notebook for Experiment 3.
2. Wear a lab coat and gloves during all procedures.
Polymerase Chain Reaction and Gel Electrophoresis 1
Part B. Dilution Calculations
1. Do this section on your own and turn-in next Wednesday, 10/17 at the
beginning of lab as homework!!! The lab late policy will apply to anything
turned in after then.
2. PCR requires two skills: calculating dilutions and pipetting small volumes. In this
section, you will do the dilution calculations.
3. The recommended PCR reagents (explained in the lecture on PCR) and their
amounts are listed in the table below:
Reagent
Up primer
Low primer
pCDNA
Taq DNA polymerase
Buffer A
dNTPs
milliQ water
Total volume
Amount Provided
1.22 nmol/l
1.54 nmol/l
985 ng/l
5000 units/ml
10X
10 mM
-----
Amount Needed
0.4 pmol/l
0.4 pmol/l
0.2 ng/l
1 unit/l
1X
200 M
25 l – (sum of the volume in l
of all other reagents)
25 l
4. For each reagent listed in the table above, your task is to calculate the volume
needed to achieve the needed amount in a total reaction volume of 25 l
following the instructions below. Use the dilution calculation procedure you
have already learned. Record your answers on a clean, lined sheet of paper.
5. Calculate how to dilute the Up primer from 1.22 nmol/l to 0.4 pmol/l. The
total volume of the diluted solution will be 25 l. Do you think you can pipette
that volume accurately? I’m guessing the answer is NO!
6. To handle this issue, we must first dilute the Up primer 1:200, and then,
calculate the volume needed from the diluted Up primer. Calculate how to dilute
the Up primer from 1.22 nmol/l to 6.1 pmol/l. The total volume of the diluted
solution will be 200 l.
7. Calculate how to dilute the diluted Up primer from 6.1 pmol/l to 0.4 pmol/l.
The total volume of the diluted solution will be 25 l. Now you should have a
volume you can pipette!
8. Calculate how to dilute the Low primer from 1.54 nmol/l to 0.4 pmol/l. The
total volume of the diluted solution will be 25 l. Do you think you can pipette
that volume accurately? I’m guessing the answer is NO!
9. To handle this issue, we must first dilute the Low primer 1:200, and then,
calculate the volume needed from the diluted Low primer. Calculate how to
Polymerase Chain Reaction and Gel Electrophoresis 2
dilute the Low primer from 1.54 nmol/l to 7.7 pmol/l. The total volume of the
diluted solution will be 200 l.
10. Calculate how to dilute the diluted Low primer from 7.7 pmol/l to 0.4 pmol/l.
The total volume of the diluted solution will be 25 l. Now you should have a
volume you can pipette!
11. Calculate how to dilute the pCDNA from 985 ng/l to 0.2 ng/l. The total volume
of the diluted solution will be 25 l. Do you think you can pipette that volume
accurately? I’m guessing the answer is NO!
12. To handle this issue, we must first dilute the pCDNA 1:100, and then, calculate
the volume needed from the diluted pCDNA. Calculate how to dilute the pCDNA
from 985 ng/l to 9.85 ng/l. The total volume of the diluted solution will be 100
l.
13. Calculate how to dilute the diluted pCDNA from 9.85 ng/l to 0.4 ng/l. The total
volume of the diluted solution will be 25 l. Now you should have a volume you
can pipette!
14. Calculate how to dilute Taq DNA polymerase from 5000 units/ml to 1 unit/l.
The total volume of the diluted solution will be 10 l.
15. Calculate how to dilute Buffer A from 10X to 1X. The total volume of the diluted
solution will be 25 l.
16. Calculate how to dilute the dNTPs from 10 mM to 200 uM. The total volume of
the diluted solution will be 25 l.
17. Add up all of the volumes you calculated above and subtract that number from
25 l. That will be the volume of milliQ water you will use for the pCDNA sample.
18. You also will run a negative control which does not contain pCDNA. Add up all of
the volumes you calculated above except for the pCDNA volume and subtract
that number from 25 l. That will be the volume of milliQ water you will use for
the negative control sample.
19. Reminder: Turn these calculations in as homework at the beginning of lab next
Wednesday, 10/17. The lab late policy will apply to anything turned in after
then.
Part C. PCR
1. Your instructor will have provided you with crushed ice in a styrofoam cooler.
2. Keep all reagents on ice while you are working.
3. Go to your storage box in Freezer -20A, and remove and place on ice the
following tubes: Buffer A, Up primer, Low primer, dNTP, pCDNA.
Polymerase Chain Reaction and Gel Electrophoresis 3
4. Obtain two 0.2ml PCR tubes, and label one as E3: pCDNA and one as E3: –CTL
(negative control). Put both tubes on ice. Once they have thawed, pipette the
following reagents into each tube in the order the reagents are listed in the
table below.
Reagent
milliQ water
Buffer A, 1X
Up primer, 0.4pmol/l
Low primer, 0.4pmol/l
dNTPs, 400M
pCDNA, 0.4ng/l
Taq DNA polymerase,
1unit/ul
Final Volume
pCDNA Tube
16.6 l
2.5 l
1.6 l
1.3 l
1 l
1 l
1 l
Instructor will add.
25 l
-CTL Tube
17.6 l
2.5 l
1.6 l
1.3 l
1 l
None!
1 l
Instructor will add.
25 l
5. After you have pipetted all reagents into both PCR tubes, keeping both tubes on
ice, bring them to your instructor who will add the Taq DNA polymerase.
6. After the instructor has added the Taq DNA polymerase, flick each tube to mix
and then, spin them briefly in the microfuge to bring the reaction volume to the
bottom of tube.
7. Place each tube in thermocycler set to the following parameters:
Initial Denature: 94ºC for 120 seconds
Denature (melt): 94ºC for 30 seconds
Anneal: 55ºC for 30 seconds
Extend: 72ºC for 60 seconds
Final Extension: 72ºC for 300 seconds
Hold: 4ºC
30-35 cycles
8. The instructor will run the thermocycler.
9. Put all of your PCR reagents back into your box in Freezer -20A.
Part D. Agarose Gel Electrophoresis of DNA
A. While your PCR samples are in the thermocycler, prepare the agarose/Syber®
Safe/TAE solution:
1. Label a 250 ml media bottle as 1.2% agarose, 1X Syber® Safe in 1X TAE buffer.
Also, label it with the date, BAMA, and your group number.
2. Weigh out 1.2 g of agarose and add it to a 500 ml graduated cylinder.
3. Add 1X TAE buffer to the cylinder bringing the total volume to 100 ml. Swirl the
solution in the cylinder gently to mix the agarose into the buffer.
4. Pour the agarose solution into the 250 ml media bottle. To make sure you have
obtained all of the agarose powder from the graduated cylinder, you may need
Polymerase Chain Reaction and Gel Electrophoresis 4
5.
6.
7.
8.
to pour the solution back and forth between the graduated cylinder and the
media bottle a few times.
Loosely cap the media bottle, and to fully dissolve the agarose, microwave the
bottle at 50% power for several seconds. Watch the bottle and stop the
microwave if the solution begins to boil over.
Using your hot hands protector, remove the media bottle from the microwave,
and swirl the solution gently. Look to see if all of the agarose crystals have
dissolved. If they haven’t fully dissolved, you will see “tails” of crystals moving in
the solution after you swirl it. If any agarose crystals are remaining, repeat the
microwave step until all crystals are completely dissolved.
Set the agarose solution on the bench and allow it to cool down.
Based on the concentration of the stock Syber® Safe, 10,000X, calculate how to
dilute it to a concentration of 1X. The total volume of the diluted solution will be 100
ml. Have the instructor check over your calculation. Then, add that volume of Syber®
Safe to the agarose solution. You can do this before the agarose solution has cooled
down. Cap the bottle and mix it gently to make sure the Syber® Safe distributes
throughout the agarose solution.
B. Prepare the gel electrophoresis apparatus and the gel:
1. Have the instructor show you how to assemble the electrophoresis box, power
supply, and dams.
2. After the agarose/Syber® Safe/TAE solution has cooled, pour it into the gel box,
add the comb (the instructor will show you how), and allow the gel to solidify.
3. After the gel has solidified (about 15 minutes), remove the dams.
4. Then, add enough 1X TAE running buffer to the chamber so the gel is completely
submerged and the fluid level reaches about ½ way up the comb spikes.
5. Gently remove the comb, pulling it straight up in one smooth motion so as not to
rip the hardened gel.
C. Prepare the DNA samples and DNA ladder:
1. Remove your PCR samples from the thermocycler and put them on ice.
2. Obtain three small squares of parafilm, and lay them out in a way to keep tract
of one as the DNA ladder, one as the –CTL and one as the pCDNA sample.
3. On the waxy side of a small square of parafilm, pipette 5 µl of loading dye (blue
juice). Pipette 5 µl of your -CTL sample directly onto the top of that sample.
Pipette the two samples up and down into the pipette tip to mix the two
samples together. Then, load the sample into your gel as described in D. below.
4. On the waxy side of a small square of parafilm, pipette 5 µl of loading dye (blue
juice). Pipette 5 µl of your pCDNA sample directly onto the top of that sample.
Pipette the two samples up and down into the pipette tip to mix the two
samples together. Then, load the sample into your gel as described in D. below.
5. On the waxy side of a small square of parafilm, pipette 5 µl of loading dye (blue
juice). Pipette 5 µl of the DNA ladder sample directly onto the top of that
Polymerase Chain Reaction and Gel Electrophoresis 5
sample. Pipette the two samples up and down into the pipette tip to mix the two
samples together. Then, load the sample into your gel as described in D. below.
6. Store your remaining PCR samples in the freezer at -20ºC.
D. Load the DNA samples and run the gel:
1. Load your DNA ladder/blue juice mixture into Lane 1 of the gel. Your instructor
will show you how to load the gel.
2. Load your -CTL/blue juice mixture into one lane of the gel being sure to note the
lane you used in your lab notebook.
3. Load your pCDNA/blue juice mixture into one lane of the gel being sure to note
the lane you used in your lab notebook.
4. Switch the power on.
5. Set the voltage to 70 volts and run the gel for approximately 30 minutes, or until
the dye front is approximately ¾ of the way down the gel.
6. Switch off the power supply.
7. Wearing gloves, remove the gel and bring it to your instructor who will help you
view it using our gel photo imaging system.
8. The instructor will give you a printout of a labeled photo of your gel. Put the
printout in your lab notebook.
9. In addition, in your notebook, record your observations by answering the
questions below:
a. Was the –CTL negative? If not, what could have caused the –CTL to have DNA
in it?
b. For the pCDNA sample, how many PCR fragment(s) did you observe on your
gel?
c. What were the approximate size(s), i.e., molecular weights of those band(s)?
d. How does this relate to what was expected for the pCDNA you amplified
(your instructor will provide you with the expected value)?
Polymerase Chain Reaction and Gel Electrophoresis 6