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Reporter: Week 9 July 10-16
From 2011.igem.org
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| + | Only one colony of tev cleavage site+GFP grew overnight. This colony was amplified through colony PCR (extension time=1:25). The PCR products were run on an agarose gel, which yielded a band just above 1000 base pairs. This matched the expected results, so the cells from this colony were grown up in culture overnight. Since only one colony grew, the assembly was repeated as a precaution. This assembly followed the same process as the one performed on Monday, July 11. | ||
| + | The promoter and RBS construct (J23100+B0034) was placed in front of the GFP+tev cleavage site, XylE+small linker and XylE+Imp linker constructs. These assemblies were performed following the same process as the assembly from Thursday, July 7. The transformations had poor time constants (less than 2.0), but the cells were plated on kanamycin resistant plates anyway. | ||
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| + | The new LacZ gene was cloned into K3 using new primers because the old primers contained a stop codon. The extension time of the LacZ PCR amplification was 1:15. This process was repeated with GusA using new primers. | ||
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| + | ==Wednesday, July 13== | ||
Revision as of 14:32, 1 August 2011
Contents |
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.
Tuesday, July 12
Only one colony of tev cleavage site+GFP grew overnight. This colony was amplified through colony PCR (extension time=1:25). The PCR products were run on an agarose gel, which yielded a band just above 1000 base pairs. This matched the expected results, so the cells from this colony were grown up in culture overnight. Since only one colony grew, the assembly was repeated as a precaution. This assembly followed the same process as the one performed on Monday, July 11.
The promoter and RBS construct (J23100+B0034) was placed in front of the GFP+tev cleavage site, XylE+small linker and XylE+Imp linker constructs. These assemblies were performed following the same process as the assembly from Thursday, July 7. The transformations had poor time constants (less than 2.0), but the cells were plated on kanamycin resistant plates anyway.
The new LacZ gene was cloned into K3 using new primers because the old primers contained a stop codon. The extension time of the LacZ PCR amplification was 1:15. This process was repeated with GusA using new primers.
