Team:Cambridge/Protocols/PCR

From 2011.igem.org

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==Polymerase Chain Reaction (PCR)==
==Polymerase Chain Reaction (PCR)==
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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).
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The goal of PCR is to amplify a section of DNA of interest for DNA analysis (e.g. gene insertion, sequencing, etc). The amplification rate is exponential.
===Theory===
===Theory===
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===Practice===
===Practice===
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The PCR reaction should contain the following reagents:
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The PCR reaction contains the following reagents:
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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.  
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Only the primers and the DNA template are specific to the reaction. The remaining reagents can be made as a 'Master Mix' in order to reduce the need to repeatedly pipette small volumes which amplify experimental error.  
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As with all enzyme containing reagents, the master mix should be kept in the freezer and small aliquots thawed when needed.   
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As with all enzyme-containing reagents, the master mix should be kept in the freezer and small aliquots thawed when required.   
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The remainder of the reaction is usually handled automatically by a PCR machine. It is common to perform an [[Team:Cambridge/Protocols/Gel_Electrophoresis | electrophoresis]] in order to separate out the correct DNA after PCR.
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The remainder of the reaction is handled automatically by a PCR machine. It is common practice to perform a [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] to extract the correct DNA after PCR.
An example of conditions we used for a PCR reaction using Phusion polymerase are as follows:
An example of conditions we used for a PCR reaction using Phusion polymerase are as follows:
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===Safety===
===Safety===
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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.
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No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, 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.
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Revision as of 11:04, 20 July 2011

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Polymerase Chain Reaction (PCR)

The goal of PCR is to amplify a section of DNA of interest for DNA analysis (e.g. gene insertion, sequencing, etc). The amplification rate is exponential.

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.

TODO: More Here!

Practice

The PCR reaction contains the following reagents:

Name Quantity
De-Ionized Water 35.5 μL
5x Buffer 10 μL
DNTPs 1 μL
Forward Primer 1 μL
Reverse Primer 1 μL
DNA Template 1 μL
Polymerase 0.5 μL

Only the primers and the DNA template are specific to the reaction. The remaining reagents can be made as a 'Master Mix' in order to reduce the need to repeatedly pipette small volumes which amplify experimental error.

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

The remainder of the reaction is handled automatically by a PCR machine. It is common practice to perform a gel electrophoresis to extract the correct DNA after PCR.

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

Cycle No. cycles Step Temp (C) Time (s)
1 1 1 98 30
2 30 1 98 10
2 55 30
3 72 2:30
3 1 1 72 5
4 50 1 4 99.59 (end)

Safety

No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, 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.



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