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Team:Hong Kong HKU/plasmidconstruction
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The cloning begins with using two PCR reactions creating the T7 promoter and the signal peptide coding region, and cloned into pET28a using a rolling assembly approach with traditional restriction-ligation cloning. The resulting pET28a containing the desired T7-Signal peptide region was confirmed by sequencing. We then moved on to construct the desired EutSMNLK gene cluster region, which was trouble as a cloning site has to be introduced between the Plac promoter and the EutS gene. Considering the timing and resources, we again used a two-step, traditional, rolling assembly approach, using BBa_K311004 as the template for PCR and pET28a as a platform vector for assembly. Finally, as we confirmed that the desired modified-EutSMNLK region was constructed using colony-PCR test (the binding region for T7Pro sequencing primer was lost in the cloning process due to limitations in restriction enzyme choices), we PCR-ed the whole desired region and ligated into the previous plasmid. The sequence of the plasmid was confirmed by sequencing. For laboratory details about the cloning methodology, please refer to the Methods and Protocols page for reference. | The cloning begins with using two PCR reactions creating the T7 promoter and the signal peptide coding region, and cloned into pET28a using a rolling assembly approach with traditional restriction-ligation cloning. The resulting pET28a containing the desired T7-Signal peptide region was confirmed by sequencing. We then moved on to construct the desired EutSMNLK gene cluster region, which was trouble as a cloning site has to be introduced between the Plac promoter and the EutS gene. Considering the timing and resources, we again used a two-step, traditional, rolling assembly approach, using BBa_K311004 as the template for PCR and pET28a as a platform vector for assembly. Finally, as we confirmed that the desired modified-EutSMNLK region was constructed using colony-PCR test (the binding region for T7Pro sequencing primer was lost in the cloning process due to limitations in restriction enzyme choices), we PCR-ed the whole desired region and ligated into the previous plasmid. The sequence of the plasmid was confirmed by sequencing. For laboratory details about the cloning methodology, please refer to the Methods and Protocols page for reference. | ||
Latest revision as of 02:58, 18 October 2014
Plasmid Construction
The cloning begins with using two PCR reactions creating the T7 promoter and the signal peptide coding region, and cloned into pET28a using a rolling assembly approach with traditional restriction-ligation cloning. The resulting pET28a containing the desired T7-Signal peptide region was confirmed by sequencing. We then moved on to construct the desired EutSMNLK gene cluster region, which was trouble as a cloning site has to be introduced between the Plac promoter and the EutS gene. Considering the timing and resources, we again used a two-step, traditional, rolling assembly approach, using BBa_K311004 as the template for PCR and pET28a as a platform vector for assembly. Finally, as we confirmed that the desired modified-EutSMNLK region was constructed using colony-PCR test (the binding region for T7Pro sequencing primer was lost in the cloning process due to limitations in restriction enzyme choices), we PCR-ed the whole desired region and ligated into the previous plasmid. The sequence of the plasmid was confirmed by sequencing. For laboratory details about the cloning methodology, please refer to the Methods and Protocols page for reference.
