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Reporter: Week 9 July 10-16
From 2011.igem.org
Sunday, July 10
The cleavage sites+GFP colonies were amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which showed that the tev cleavage site+GFP colony was vector background. The cI cleavage site+GFP contained the correct number of base pairs, so the colony was grown in culture overnight. The sequencing results (which came back on Monday, July 11) showed that the cI cleavage site did not successfully assemble to the GFP vector.
The promoter and RBS construct (J23100+B0034) was placed in front of the GFP+cI cleavage site and in front of the XylE+small linker construct. The same assembly process from Thursday, July 7 was repeated for this assembly.
Monday, July 11
The assembly 25 (openwetware) method for fusion parts was used to place the tev cleavage site in front of GFP. The tev cleavage site insert was cut with EcoRI and AgeI in buffer 1. The GFP vector was cut with EcoRI and NgoMIV in buffer 4. The insert and vector were ligated together, transformed into electrocompetent Escherichia coli cells then plated onto kanamycin resistant plates.
