Polymerase Chain Reaction (PCR) DNA Amplification
To learn the principles and techniques of DNA amplification via polymerase chain
The polymerase chain reaction (PCR) assay is one of the foundations for research and
biotechnology. Scientists are applying the benefits of this assay in the research laboratory
as well as in the areas of pharmaceutical development, forensic investigation, agricultural
management, and more. PCR allows exponential amplification of specific genes through
a series of controlled temperature changes. The investigator must know the genetic
sequence surrounding the gene of interest so the proper primers may be added to the
reaction. A primer is a known sequence of DNA usually 15 to 24 base pairs in length.
The primers will anneal to the DNA flanking the gene of interest. DNA polymerase is
an enzyme that forms the replication machinery and must also be added for extension of
the flanking primers. Free nucleotides are added so the enzyme can lay bases down. The
reaction is heated causing the double stranded DNA to denature or separate into single
stranded DNA. The temperature is then decreased to allow the primers to anneal to the
single stranded DNA at their complimentary loci. The replication machinery extends the
new complimentary strand of DNA leading to double stranded DNA. The reaction is
then heated once again to denature the double stranded DNA and the process is repeated
for 30 to 35 cycles.
Gloves Takara Enzyme (red)
Microcentrifugetube Rack Buffer Solution (yellow)
5 Empty Micrcentrifuge Tubes Pa Forward Primer (orange)
0.5-10 uL Micropipettor Ph Reverse Primer (orange)
20-200 uL Micropipettor Sterile Water (blue)
Sample DNA (green) Nucleotide Solution (purple)
3 PCR Reaction Tubes
Part A: Preparing the Primer Dilution
1. Label 2 empty microcentrifuge tubes with the corresponding primer name (Pa and Ph).
Make a 1:10 dilution by adding 18 uL sterile water to each tube. Allow the primers to
thaw completely and add 2 uL Pa primer to the tube labeled Pa, and 2 uL PH primer to
the tube labeled Ph.
2. Label 2 empty microcentrifuge tubes with the corresponding primer name (Pa and Ph)
and the number 50. Make a 50 concentration of each primer by adding the following
volumes of diluted primer and water to the empty tubes:
Add 11.5 uL of Pa primer to 38.5 uL sterile water
Add 13.4 uL of Ph primer to 36.6 uL sterile water
Part B: Calculate the Volume of Reagents
Before beginning a PCR reaction the volume of reagents for the reaction must be
calculated. You must count how many reactions you need to run (3 in this case) and add
1 additional reaction to your count. By doing this you will be sure to have more than
enough volume to complete the reaction.
Takara Enzyme: 0.25 uL X 4 samples = 1 uL
Buffer: 5 uL X 4 samples = 20 uL
Nucleotide: 4 uL X 4 samples = 16 uL
Pa: 2.5 uL X 4 samples = 10 uL
Ph: 2.5 uL X 4 samples = 10 uL
Sample: 5 uL in corresponding reaction tube
Sterile Water: 19.25-|50|= 30.75 X 4 samples = 123 uL
(To obtain the value for sterile water all other volumes must be added up and then
subtracted by 50 since each of the three reaction tubes will be completed to a volume of
50 at the end of the assay. That value must then be multiplied by the number of reactions
taking place (3 actual reactions plus 1 extra).
Part C: PCR Amplification
1. Place the 3 PCR reaction tubes containing the sample DNA and 3 empty
microcentrifuge tubes in a microcentrifuge rack. Label the empty tubes to correspond
with the sample DNA.
2. Add 5 uL sample to each corresponding tube.
3. Label the final empty micrcentrifuge “MM” for Master Mix.
4. Add 123 uL sterile water to the MM tube.
5. Add 20 uL buffer to the MM tube.
6. Add 16 uL nucleotide solution to the MM tube.
7. Add 10 uL of each primer to the MM tube.
8. Add 1 uL Takara enzyme to the MM tube.
9. Gently tap to mix. Do not invert.
10. Add 45 uL Master Mix solution to each of the reaction tubes.