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Team:Freiburg/Notebook/Methoden
From 2012.igem.org
Methods
PCR Methods
In this protocol you can find the ingeredients and the thermocycler program for a working extension PCR. If you are thinking about doing a own extension PCR, you need to set the reaction conditions according to your experiment design. This means to adjust annealing temperature for your primers as well as the extension time for the lenght of your template. You can find instructions inside the manual of your polymerase on how to do this ajustments.
As you can see, our PCR program has two-steps, the first step is to extend the template and the second to amplificate the now longer template. The two steps are neccesary due to the different annealing conditions for extension and amplification. During extensions only a short part of the primer binds to the template which needs a lower temperature, at the amplification step the complete primer binds so you need a higher temperature.
This protocol is essentially the same as the first extension PCR. It is just here for completeness
Colony PCR is one of the standard methods in molecular biology and you need it at almost every project. Essentally you pick a colony after transfection put a bit of it into a PCR tube and use it as template for a PCR. The PCR amplifies the part between the primers and you should be able to make it visible on an agarose gel. Together with a gel ladder you can now tell the lenght of the amplificated part and with this make suggestions if your transfection worked out.
Purification, Digest and Ligation
In this protocol we combined all of the small processes we used during our project, for most of them like PCR purification or plasmid preparation we used commercial kits. These kits come with easy step by step explanations and do not need much practice. Even people that cannot be in the lab every day can do them without worrying about destroying precious samples.
Transformation and Transfection
Transformation is a thing in the lab for which everyone has a little trick to do it best and everybody tells you that their way is the right way because they do it since forever. As we can tell you, our protocol is perfectly working, its effective and gives you great transformation efficiency if you use it like it is written.
Transfection of HEK cells is almost as easy as transforming E.coli. With this protocol we got great transfection efficiency - even when we used multiple plasmids. Please make sure you thought about your experiment design! We would advise to always do three replicates and implement multiple control experiments. This is especially important when you do co-transfections of multiple plasmids.
