Sean Kelley

Formal Instruction Assignment

March 1st, 2016

Instructions on performing a basic polymerase chain reaction.

Polymerase chain reactions (PCR) are used in the field of molecular biology to
replicate strands of DNA and make it easier for forensic scientists to observe small
samples of DNA. In these instructions the process of PCR will be explained and
every step in the process is surprisingly simple to understand.
The main cause of a failure of replication in the experiment will be due to
contamination of the DNA, nucleotides and primers. In order to avoid contamination,
the buffer solution where the DNA sample is held must be kept in a sterile
environment and not in contact with humidity and harsh light. Failure of this process
in the field of forensic sciences can be devastating to an investigation as the sample
will be useless if contaminated.
In order to replicate your DNA through PCR, you will need a few instruments. A DNA
template to amplify, nucleotides containing triphosphate groups, Taq polymerase, 2
three prime primers, dNTPs, a thermal cycler and a buffer solution to keep your DNA
in a suitable environment.
1. Place the thermal cycler in a cool, dry and sterile environment. Use
antibacterial wipes in and around the cycler while it is powered off and
unplugged. This cleaning will help prevent contamination of the DNA and
ensure the success of the process.
2. In order to unwind the double helix of DNA, the sample is placed in capsuleshaped test tubes in the thermal cycler at 94 degrees Celsius to denature the
hydrogen bonds of the DNA and produce single-stranded templates. Later,
these single strands will be have complementary bases filled in to complete
several double helix strands.
3. The sample now needs its primers to begin the annealing stage, so the
primer must be injected into the DNA sample and placed back into the
thermal cycler. The cyclers temperature must be lowered to 5565 C for 20
40 seconds allowing the primers to anneal to the single-stranded DNA
template. However, if the temperature is dropped too low during the
annealing stage, the primers will bond with the wrong complementary bases
and nullify the experiment. Make sure to observe the temperature during the
annealing stage as it will be used later.
4. In our experiment the Taq polymerase will be kept at 7580 C in the cycler.
At this step the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding dNTPs that are
complementary to the template under the right conditions and if the

experiment was conducted successfully, the growth of the DNA sample will
become exponential and available for further for observation.
5. After the polymerase begins this exponential multiplication of DNA, there will
be three stages associated with the production of DNAs decline. The
exponential growth, levelling off of growth where the amount of reagents
becomes limited and replication slows down and plateau where the
replication ceases. The temperature that was taken during the annealing
stage will help compute the time left before the replication ceases if it is
utilized in a rate of change equation.
6. By now you should have thousands of copies of your sample DNA and the
PCR process was a success.