From 2010.igem.org
Duke iGEM 2010 Notebook
===10/26/10:===
Biobricks K429000 and K429001 submitted to registry.
===10/25/10:===
Samples of BioBrick submissions K429000 and K429001 were successfully miniprepped and were packaged for shipping.
CPEC reaction from 10/24/10 showed only faint banding, so the extension cycle duration was increased and the reaction was performed again.
===10/24/10:===
BioBricks from 10/23/10 were transformed into GC5 and grown in liquid medium.
First attempt at CPEC was performed.
===10/23/10:===
K429000 and K429001 samples from 10/22/10 were miniprepped and cut with EcoRI and PstI, the ligated into the precut linearized pSB1C3 plasmids supplied by the registry.
PCR of plasmid DNA from 10/22/10 produced insufficient yield of the fully extended plasmid, so another run of PCR was performed on the sample with varied annealing temperatures to find an optimal annealing temperature.
===10/22/10:===
Q04400, Q04510 and I13504 were first miniprepped, then digested and ligated following the standard assembly process. Gel electrophoresis was performed on the samples and they appeared to have correctly ligated, so they were then transformed into GC5 colonies and grown in a liquid medium.
PCR assembly of [J23100 + P0412 + B0015 + pSB1C3 overlap] appeared to be successful; the product was extracted from the gel and further extended by the PCA of [pSB1C3 overlap + pLacI +RBS], so that the pSB1C3 plasmid would be ready for CPEC.
===10/18/10:===
PCR assembly of [J23100 + P0412 + B0015 + pSB1C3 overlap] was unsuccessful; annealing temperatures were changed and the reactions were run again. All other PCRs from 10/17/10 were successful, and upon gel extraction the samples were prepared for the CPEC. Noting that problems arising with CPEC would mean that we would be unable to submit BioBricks by the deadline, parts Q04400, Q04510 and I13504 were transformed into GC5 and grown in liquid cultures for extraction.
===10/17/10:===
PCA of [A-fos + B0015], [J23100 + RBS + c-fos] were successful, and were followed by the PCA of the new segments. Each PCA product was run through a 1% agarose gel; banding was very faint for the final product, and upon gel extraction only .056ug of the product DNA were yielded. This product was put through another round of PCR prior to the second assembly step, and upon gel extraction the product yield was of .218ug. The PCR assemblies for [c-jun + B0015 + custom promoter], [RBS + tetR + B0015 + RBS + GFP + terminator], [J23100 + P0412 + B0015 + pSB1C3 overlap] and [pSB1C3 overlap + R0010 +RBS] were started.
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