Difference between revisions of "Part:BBa K496000"

Latest revision as of 15:11, 1 October 2011

Forward primer for visualizing restriction enzyme digestion.

Fig. 1

Fig. 2

   Sometimes standard protocol fails to produce plasmid, or BioBrick is toxic to host cell and can't be 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 we amplify BioBrick parts or vectors, primers which anneal to prefix and suffix are used, because they are universal. But unlike miniprep products, PCR products don't produce two distinct bands when they are digested. So we are left wondering if digestion went well.

   The iGEM HokkaidoU 2010 created a set of primers which solves this problem. The idea is quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the Biobrick (Fig. 1).

   PCR products that are amplified by using this primer set produces distinct approx 100 bp and 200 bp fragments (Fig. 2).

   You can judge if digestion was successful by presence of these bands. Compared to the intact PCR product, you will see the significant change in digested PCR fragment length. The fact that all 10 current high copy number assembly vectors containing pSB1C3 share the same annealing sites in the BioBrick flanking region makes this possible.

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

Sequence and Features