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Polymerase chain Reaction
PCR is a rapid, inexpensive and simple way of amplifying specific DNA fragments from
minute quantities of source material. This revolutionary method was developed by Kary
Mullis in the 1980s. PCR methodology employs a temperature resistant polymerase enzyme,
which works in the presence of a short stretch of single stranded DNA called primer by consuming
the substrate dNTPs and finally amplifies the template DNA specifically. As the name
indicates, PCR is a chain reaction having repeated cycles, each of which consists of
three steps: Denaturation, Annealing, and Extension. Denaturation is achieved with an
increase in temperature to 100 degree centigrade, primer annealing at 54 degree centigrade and
extension is achived by Taq polymerase at 72 degree centigrade. Repeated heating and
cooling cycles which is accomplished by the thermal cycler amplify the target DNA exponentially.
20 PCR cycles can amplify the target by a million fold.
Materials Required
Icebox RNase Free DNase Free distilled water
10x buffer Plasmid template
Forward primer Reverse primer
dNTPs Taq polymerase
PCR tube Micro Pipettes
Pipette tips Thermocycler
Procedure
Arrange all tubes in the ice box. Take a 10-100 µl pipette and adjust the volume
to 37.5µl. From the tube placed in the ice box pipette
out RNase Free DNase Free distilled water. Add this solution into a 0.5 ml PCR tube taken
from the ice box. Place the PCR tube back in the ice box.
Discard the used tip. Now take 0.5-10 µl pipette and adjust the
volume to 5 µl. From the tube placed in the ice box, pipette
out 10x buffer. Add this solution into the same PCR tube.
Place the PCR tube back in the ice box. Discard the used tip.
Prepare the reaction mix by adding the following components in the respective volume: 1 µl
of plasmid template, 2 µl of dNTPs, 2 µl of forward primer, 2 µl of reverse primer
and 5 µl of taq polymerase, using the same PCR tube.
Mix the solution in the vial using the pipette. However ensure you do not mix this vigorously,
as this will inactivate the enzyme taq polymerase) Turn on the Thermocycler
Place the PCR tube containing the Reaction mix in the PCR machine.
Close the lid and set the specific PCR program. Then press the start button to run the PCR
program.
Result:
The amplicons which are formed after the specific amplification of the target sequence can be
detected using the technique of gel electrophoresis.