Team:Grenoble/Biology/Protocols/PCR 1
From 2012.igem.org
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<li>Set up the thermocycler at the right temperatures.</li> | <li>Set up the thermocycler at the right temperatures.</li> | ||
<br/> | <br/> | ||
- | <li>In a | + | <li>In a 200 µL PCR tube, introduce : <ul><div class="petit"> |
<li>1.25 µL of forward primer (10µM)</li> | <li>1.25 µL of forward primer (10µM)</li> | ||
<li>1.25 µL of reverse primer (10µM)</li> | <li>1.25 µL of reverse primer (10µM)</li> | ||
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</center> | </center> | ||
<br/> | <br/> | ||
- | <li>Put the | + | <li>Put the 200 µL PCR tube into the thermocycler.</li> |
<br/> | <br/> | ||
<li>Turn on the device.</li> | <li>Turn on the device.</li> |
Revision as of 14:47, 3 September 2012
PCR using a GoTaq® polymerase
Goal
Amplify a section of DNA.Protocol
Following are the instructions from the Promega website.- Set up the thermocycler at the right temperatures.
- In a 200 µL PCR tube, introduce :
- 1.25 µL of forward primer (10µM)
- 1.25 µL of reverse primer (10µM)
- 2.5 µL of dNTP (2 mM)
- 5 µL of GoTaq® Reaction Buffer
- variable volume of template DNA
- 0.12 µL of GoTaq® polymerase
- to 25 µL of nuclease-free water
SAFETY AND USEFUL RECOMMANDATION Those products are not dangerous at first sight, but in case of spreading or contacts with any part of the body, it is recommended to wash this part and to dry it after. Use a different pipette tip for each sample. It is also important to close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.
- Put the 200 µL PCR tube into the thermocycler.
- Turn on the device.
It was noticed that it exists a certain number of different thermocycler models.
That is why biologists do not know, all the time, how the device they will use, has to be manipulated. This
can cause risks for thermocycler users. Indeed a thermocycler is an electric device which can become hot,
and in case of misused people can be injured. Consequently it is very important for users to know how to use
the device. It is recommended to read the operating instruction before using it. In our group we managed to
learn how to use it before doing any manipulation. Besides on the device there are also protections to prevent
accident. |