LAB 5
POLYMERASE CHAIN REACTION
Polymerase chain reaction (PCR) refers to a technique that amplifies one copy
of DNA into billions of copies (of the same DNA) within a few hours. PCR uses a
thermostable DNA polymerase enzyme to amplify a piece of DNA known as the
template DNA. DNA polymerase can build new DNA using the nucleotide
sequence of the template DNA, provided that an oligonucleotide primer (or a
short piece of DNA) is bound to the template. Primer provides a starting point for
DNA polymerase to add nucleotides for the synthesis of new DNA. The building
blocks of DNA, four individual nucleotide triphosphates (dNTPs), should be
provided in the reaction. Unlike ordinary DNA polymerase, thermostable DNA
polymerase is not inactivated by heating up to 95º C, because the thermostable
enzyme is extracted from organisms that live in extreme hot environments such
as hot-springs and deep sea vents. For example, Taq DNA polymerase is
extracted from the hot springs bacterium Thermus aquaticus. The polymerase
enzyme buffer provides the necessary salt, pH, and MgCl2 required for optimum
enzyme function. If MgCl2 is not present in the enzyme buffer, it must be added
separately.
PCR process requires repeated cycles of DNA denaturation (separation of double
strands of template DNA), primer annealing (binding of primers to each single
strand of DNA), and extension (synthesis of new DNA). In each cycle, the number
of DNA copies doubles. By selecting appropriate primers or starting points, PCR
process can be designed to amplify only a certain segment of a gene starting from
an organism’s genomic DNA. Widely practiced throughout molecular biology
laboratories, PCR has numerous uses such as identification of organisms,
identification of genetic susceptibilities and forensics.
This exercise is designed to help you become familiar with the PCR technique. We
will amplify a bacteriophage lambda (λ) DNA fragment of 1.1 kb. The nucleotide
sequence of all 48,502 base pairs of λ DNA is known. (This 1.1 kb fragment
contains the 784 bp λ BamH1/HindIII double digest). The forward primer for the λ
1.1 kb fragment has the sequence 5’ TTCTGAACTCGGTCCGTTAC 3’
corresponding to nucleotide numbers 22,233 to 22,252 of λ DNA and the reverse
primer is 5’ TCGCCAACATCATTCGACTC 3’ from nucleotides 23,320 to 23,339. λ
DNA will be amplified by PCR using these specific primers. We will then perform
agarose gel electrophoresis of the PCR product. This will allow us to determine
whether the targeted λ fragment has been amplified by comparing its size to a
known DNA size marker or λ BamH1/HindIII double digest.
PCR AMPLIFICATION OF 1.1 KB λ DNA FRAGMENT
Materials needed:
Thin walled 0.5 ml microfuge tubes
Microfuge tube racks
Ice in containers
Thermal cycler
Taq DNA polymerase (5U/µl) (keep Taq on ice)
5X PCR buffer (containing 15 mM MgCl2)
Sterile water
Forward and reverse primers: 5 µM
Template: λ DNA (1.0 ng/µl)
dNTPs: 2.5 mM (diluted from 100 mM stock)
Sterile MgCl2 (25 mM)
Method:
1. Label tops of seven 0.5 ml microfuge tubes with initials of your name and
your partner’s name. Label first six tubes with the tube number (1-6).
Label the seventh tube as 7X. Tubes 1 and 2 are single primer controls
and tube 3 is a no template control. Tubes 4, 5 and 6 get both primers but
will have varying amounts of the template. The seventh tube will be used
to prepare a master mix of 7X.
2. Prepare a master mix of 7X to be used for the six tubes. Master mixes are
prepared when we have to mix small volumes of common reagents that
will be added to all the tubes. We only need amounts for six tubes, but the
master mix will be prepared for quantities calculated for seven tubes
(hence 7X) to allow for pipetting errors and make sure you will have
sufficient quantities for the six tubes.
3. Add the following reagents to the master mix (7X) tube placed on ice. Only
those reagents that are common to all six tubes will be added to the
master mix. Reagents that are not common to all tubes will be omitted
from the master mix and added to the individual tube. Add the components
in the order listed. (1X values are given for your information only)
Water
dNTPs (2.5 mM)
5X Buffer (green)
MgCl2 (25 mM)
Taq (5 U/ µl)
Total
1X
12.375 µl
2.5
µl
5.0
µl
2.0
µl
0.125 µl
22.0 µl
7X
86.63 µl
17.5 µl
35.0 µl
14.0 µl
0.87 µl
154.0 µl/7= 22 µl
4. Close 7X tube and mix well by flicking and turning the tube upside down.
Spin briefly in a microfuge. Aliquot 22.0 µl to tubes 1-6.
5. Place all tubes on ice. Add forward primer, reverse primers and varying
levels of template to the specific tubes as given below.
1
2
3
Master mix
Forward primer
Reverse primer
λ DNA (1ng/µl)
Water
22.0
---1.0
1.0
1.0
22.0
1.0
---1.0
1.0
22.0
1.0
1.0
---1.0
Total
25.0
25.0
25.0
4
5
6
22.0
1.0
1.0
0.5
0.5
22.0
1.0
1.0
1.0
----
22.0
1.0
1.0
1.5
----
25.0
25.0
25.5 µl
µl
µl
µl
µl
µl
6. Close all tubes. Each tube should contain about the same volume. Gently
tap the tubes to bring liquid to the bottom of tube. Place in a microfuge
and spin with a short pulse.
7. Place tubes in the thermal cycler. Start the pre-programmed file. Thermal
cycler will run the following:
One time initial denaturation: 3 min at 95o C
20 cycles of
 Denaturation: 1 min at 95o C
 Primer annealing: 30 seconds at 52o C
 Extension: 1 min at 72o C
One time final extension: 7 min at 72o C
The block will cool down to 4o C
AGAROSE GEL ELECTROPHORESIS (Review information in previous lab)
In order to visualize the amplified DNA, PCR products are separated by agarose
gel electrophoresis and stained with ethidium bromide, The stained gel is
observed under UV light. In this lab we will use 1.25% agarose instead of the
0.8% used in the restriction digestion previously.
Materials needed:
Agarose
Balance
Erlenmeyer flasks
1X TBE buffer
Mittens
Gel plates with combs
Bubble level
Gel boxes
Power supply
Gel loading buffer
Microwave
100 bp DNA size marker ladder
Method:
1. Prepare 100 ml of a 1.25% agarose solution in 1X TBE buffer in the
Erlenmeyer flask. Weigh the flask with buffer and agarose and write down
in a piece of paper. Heat in the microwave on LOW/MEDIUM POWER in
short pulses to dissolve agarose. CAUTION: IF HIGH POWER IS USED
AGAROSE CAN BOIL OVER. USE MITTENS TO REMOVE THE FLASK
FROM THE MICROWAVE. Weigh the flask with contents again after
microwaving and add back the water lost. Let cool for 5-10 mins.
2. Cast agarose gel: Place a mini gel plate inside a locking-plate with a
rubber seal, as demonstrated by the instructor. Press the mini gel plate
firmly on to the locking plate. Locking plate will prevent agarose from
leaking from the two open sides of the mini gel plate. Place a comb about
1 cm from one end of the mini gel plate. Teeth of comb should not touch
the bottom of wells. Find a level bench to pour the gel. You can use the
leveling bubble for this purpose. Pour melted agarose solution and let
cool. Agarose will solidify as it cools. Once solidified, remove the comb
and the locking plate with rubber seal.
3. Add 1X TBE to the gel box. Place agarose gel with plate in the gel box. If
necessary add more 1X TBE to cover the gel.
4. Add DNA ladder to the first well to use as a DNA size marker. First add the
loading dye to the DNA ladder by mixing 25 µl of DNA ladder stock tube
and 5 µl of the 6X loading dye to get a total of 30 µl. Load 8 µl of this to the
first well. Hold the micropipet upright, lower the tip into the well and
slowly dispense sample into the well.
5. Starting from the second well, load 10 µl of the PCR amplified tubes 1-6,
one sample per well. If a colored PCR buffer or enzyme was used there is
no need to add loading dye separately to the PCR tubes. (For example,
Promega’s Go Taq 5X buffer contains a green dye. This green dye
consists of two different dyes, blue and yellow, that separate during
electrophoresis. Blue dye is heavier and runs with 3-5 kb DNA fragments,
whereas the yellow dye is lighter and runs with ~500 bp DNA fragments.
Blue and yellow dyes together appear as green).
6. Save the rest of the PCR product to be used in Lab 6 for cloning.
7. After all the samples have been loaded, cover the gel box with lid and
connect electrical leads to the power supply. Be sure to connect the
electrical leads to the power supply properly. ELECTRICAL LEAD FROM
THE TOP OF THE GEL BOX (START POINT) CONNECTS TO THE
NEGATIVE IN THE POWER SUPPLY AND THE LEAD FROM THE BOTTOM
OF THE GEL BOX (END POINT) CONNECTS TO THE POSITIVE IN THE
POWER SUPPLY. Turn the power supply on and adjust the voltage to get
70-80 V.
8. Do you see the bubbles form? Bubbling is another way to confirm that the
electricity is flowing through the gel. Let the gel run until the blue band of
the loading dye has run 2-3 cm from the end of the gel.
9. Turn the power off and remove the gel plate from the gel box. Do not
remove gel from the plate. Proceed to staining.
GEL STAINING AND DOCUMENTATION
(Review information from previous lab)
RESULTS:
1. Examine your gel photograph. Has the gel run, stained and destained
properly? Do you see clear bands in the DNA size marker ladder in the
first lane? If you can see the DNA ladder ok, then your gel has run
properly.
2. Do you see any bands in lanes 2, 3, 4, 5, 6 and 7 that correspond to PCR
tubes 1 through 6? If you do observe bands, determine their size using the
marker DNA ladder.
3. Can you interpret your results?
Result:
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Interpretation: