PCR using a HF Phusion polymerase

Goal

Protocol

1. Set up the thermocycler at the right temperatures.


2. In a 50 µL PCR tube, introduce :
   1.25 µL of forward primer
   1.25 µL of reverse primer
   2.5 µL of dNTP (2 mM)
   5 µL of buffer
   0.75 µL of DMSO (optional)
   variable volume of template DNA
   0.25 µL of HF Phusion 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 in case of products on hands or on any parts on the body, it is recommended to wash this part and to dry it after. Be careful to use for each sample a different sterile cone. It is also important to well close tubes after putting the ingredient in. Moreover the DNA cannot resist until it is not incorporated in a plasmid and into a cell. Therefore there is no chance that there are any possible consequences because of a leakage in the environment.

   
   
3. Put the 50 µL PCR tube into the thermocycler.


4. Turn on the device.



SAFETY AND USEFUL RECOMMANDATION

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.