Difference between revisions of "Part:BBa K496000"

Revision as of 03:37, 28 October 2010

Forward primer for visualizing restriction enzyme digestion.

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 primers which anneal 100 bp upstream and 200 bp down stream from the part like in Fig.1 bellow.

Fig.2

Now when digested PCRed fragment produces distinct approx 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 Fig.2). This is crucial when you take into account that many participants have limited at the bench experience before hand.

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 up to about 10.

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

This primer set makes amplified BioBricks 300bp longer making small parts like RBS easier to extract from gel. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.

Works with With assembly plasmids pSB1A3 pSB1A7 pSB1AC3 pSB1AK3 pSB1AT3 pSB1C3 pSB1K3 pSB1T3

Biobricks PCRed with these primers produce fragment approximatively 100 bp and/or 200bp when digested with restriction enzymes.

Fragment lenght

Does not work with, because one of the primers site is missing:
pSB2K3

Sequence and Features