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Team:Yale/Our Project/Methods/
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| - | + | The experimental methods used for generation of plasmid constructs containing IGEM parts registry promoter (insert name here) as well as the phsABC thiosulfate reductase gene and the terminator are described as follows. The vector containing phsABC was provided to us by the ?Keasling Lab?. This vector underwent two successive single digestions in which each digestion was allowed to proceed overnight. This deviation from the standard double digestion was necessary to insure successful cutting of the phsABC insert out of the provided plasmid. Following these digestions, the DNA components in the digestion reaction solution were analyzed via gel electrophoresis and the dna band that corresponded to the length of the phsABC insert was excised from the gel and gel purified. Plasmid pSB74!!!!!!?-is this the igem one? or the one we recieved?!!!!!!!!!!!, which has been shown to posses the highest activity, was also digested and purified in a manner similar to what is described above for the insert. These purified dna segments were then combined in a ligation reaction that used T4 dna ligase to combine the sticky ends of the degenerate restriction sites, allowing for the formation of phsABC insert containing pSB74 plasmid. The resultant plasmid was sequenced and then transformed to increase its concentration. The transformed cells were then miniprepped using the Qiagen miniprep system. (sequencing) (resistance) This plasmid then underwent an very similar series of steps to insert the promoter and regulatory biobricks. | |
<!------------- METHODS: NEEDS TO BE EDITED -------------> | <!------------- METHODS: NEEDS TO BE EDITED -------------> | ||
Revision as of 06:06, 27 October 2010
our project
experimental methods
The experimental methods used for generation of plasmid constructs containing IGEM parts registry promoter (insert name here) as well as the phsABC thiosulfate reductase gene and the terminator are described as follows. The vector containing phsABC was provided to us by the ?Keasling Lab?. This vector underwent two successive single digestions in which each digestion was allowed to proceed overnight. This deviation from the standard double digestion was necessary to insure successful cutting of the phsABC insert out of the provided plasmid. Following these digestions, the DNA components in the digestion reaction solution were analyzed via gel electrophoresis and the dna band that corresponded to the length of the phsABC insert was excised from the gel and gel purified. Plasmid pSB74!!!!!!?-is this the igem one? or the one we recieved?!!!!!!!!!!!, which has been shown to posses the highest activity, was also digested and purified in a manner similar to what is described above for the insert. These purified dna segments were then combined in a ligation reaction that used T4 dna ligase to combine the sticky ends of the degenerate restriction sites, allowing for the formation of phsABC insert containing pSB74 plasmid. The resultant plasmid was sequenced and then transformed to increase its concentration. The transformed cells were then miniprepped using the Qiagen miniprep system. (sequencing) (resistance) This plasmid then underwent an very similar series of steps to insert the promoter and regulatory biobricks.
