Team:Cambridge/Protocols/PCR

Polymerase Chain Reaction (PCR)

The goal of the PCR protocol is to amplify a section of DNA which is of interest in order to carry out some operations on the section (e.g. insertion into a plasmid, sequencing, etc). PCR allows the amplification of a section of a single molecule into a large number of identical molecules (~10^9).

Theory

Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses. Credit, WikiMedia

The reaction works by the annealing of primers to single stranded DNA, which are extended by DNA polymerase. By repeating the process, the section of DNA between the primers is amplified.

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Practice

The PCR reaction should contain the following reagents:

Only the primers and the DNA template are specific to the reaction, and thus the remaining reagents can be made as a 'Master Mix' in order to reduce the need to repeatedly measure small volumes for each tube.

As with all enzyme containing reagents, the master mix should be kept in the freezer and small aliquots thawed when needed.

The remainder of the reaction is usually handled automatically by a PCR machine. It is common to perform an electrophoresis in order to separate out the correct DNA after PCR.

An example of conditions we used for a PCR reaction using Phusion polymerase are as follows:

Safety

No bacteria are used during the reaction, and this there is little or no biological hazard. However, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature, and this may present a risk, depending on the PCR machine employed.