Team:HokkaidoU Japan/Projects/PCR

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 unlike miniprep product BioBrick amplified like this don't produce 2 distinct bands when digested. So you are left wondering if digestion went well.

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

So idea behind this primers 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 (see Fig2). This is crucial when you take into account that many participants have limited at the bench experience before hand.

Fig2

As everyone have guest by now these primers anneal to plasmid regions flanking the BioBrick. And there are more than 40 different primers. But not all of them are quite so different in fact all 7 registries assembly plasmids share the same sequence on the BioBrick flanking regions. And few others do also, bringing the number to about 10.

And with the new registry requirement to submit all BioBric in pSB1C3 plasmid present the possibility that in near future that many if not all plasmids could be supported by this primers making assembly easier.