Team:HokkaidoU Japan/Projects/PCR

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PCR Based Assembly Protocol


Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't by amplified. In moments like these PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail :).


When amplifying BioBricks primers which anneal to prefix and suffix are used. Good thing about them is that they are universal so with only 2 you can amplify all BioBricks. But because unlike miniprep product BioBrick amplified like this don't produce 2 distinct bands. So you are left wondering if digestion went well.


We created a set of primers which solve this problem. You can judge if digestion was successful by bands approximatively 100 bp or/and 200 bp bands presence and significant change in PCRed fragments length.


So idea behind this quite simple. We just designed which anneal 100 bp upstream and 200 bp down stream from the part like in figure bellow.


FIGURE


Now when digested PCRed fragment produces distinct aprox 100 bp or/and 200 bp fragment depending on cut sites. Knowing this you can continue to the next step with the sure and light heart. This is crucial when you take into account that many participants have limited at the bench experience before hand.