From 2007.igem.org

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Contents 1 Aug 2007: Order ComP and ComA from GENEART 2 Aug 2007: Primer design for PCR stitching 3 Sep 2007: 3.1 First round PCR 3.2 Second round PCR

Aug 2007: Order ComP and ComA from GENEART

We were counting on [http://partsregistry.org/Part:BBa_J51000 ComP] and [http://partsregistry.org/Part:BBa_J51001 ComA] being available in the 2007 iGEM distribution. As this was not the case, and we had DNA synthesis bases to burn, we ended up ordering them from GENEART. The original sequences, as described in the above links, were optimized for expression in e.coli and all cis-acting sites and assembly restriction sites were removed. We also included biobrick prefix/suffix and vf2 and vr primer binding sites. The two sequences, now labeled ComA' and ComP' took around 3 weeks after confirmation of order to be dispatched. The sequences have been uploaded below:

ComA' sequence (As revised by geneart)

ComP' sequence (As revised by geneart)

Aug 2007: Primer design for PCR stitching

In order to stitch NpSopII-NpHtrII to ComP at the sites detailed here, these primers were designed. We aimed to have at least 18bp on each half of the stitch, while adhering to the standard primer design guidelines.

Sep 2007:

First round PCR

Two separate PCR reactions for each construct. One to amplify the NpSopII-NpHtrII half and the other to amplify the kinase domain of ComP. Detailed protocol. Gel purification of ~1.2kb band from both reactions. Some non-specific bands observed in some reactions.

Second round PCR

Gel purified products from first round used as templates/primers in the second round PCR. Products were quite smeared, but we excised a band of ~2.5kb and gel purified.