Team:Wisconsin-Madison/parts 3

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       <p class = "classtheoverview"> <strong>Taq Ligase</strong></p>
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       <p class = "classtheoverview"> <strong>Text Plasmids</strong></p>
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       <p align="left" class = "classtheinlinecontent2">Taq Ligase is a thermostable DNA ligase, which joins adjacent 5' and 3' ends of DNA, removing nicks in double stranded DNA, active from 45-65 degrees Celsius(Barany, F. (1991). Proc. Natl. Acad. Sci. USA. 88, 189-193). The Taq ligase gene is within a kanamycin resistant PET-ALM1 plasmid, downstream of the T7 promoter, upstream of the T7 terminator. Thus, it can be used with an inducible T7 expression system to express the enzyme. The protein has an N-Terminus hexa-histidine tag, joined to the wild type structure by a thrombin cleavage motif. </p>
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       <p align="left" class = "classtheinlinecontent2">In addition to the Gibson enzyme plasmids, we have also provided test plasmids that can be used to test a gibson reaction. The product test plasmid is a UV green fluorescent protein from a p70RG plasmid inserted in a pMSBmCherry (RFP) plasmid backbone. The two parts can be amplified by PCR from their respective plasmids and then used in a gibson assembly reaction. Colonies with the PCR products will be UV Green fluorescent when induced with IPTG if the assembly is correct and false positives will have much lower UV fluorescence, the p70RG false positives having no color and the pMSBmCherry false positives being noticeably red.</p>
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Latest revision as of 21:47, 23 September 2013


Text Plasmids

In addition to the Gibson enzyme plasmids, we have also provided test plasmids that can be used to test a gibson reaction. The product test plasmid is a UV green fluorescent protein from a p70RG plasmid inserted in a pMSBmCherry (RFP) plasmid backbone. The two parts can be amplified by PCR from their respective plasmids and then used in a gibson assembly reaction. Colonies with the PCR products will be UV Green fluorescent when induced with IPTG if the assembly is correct and false positives will have much lower UV fluorescence, the p70RG false positives having no color and the pMSBmCherry false positives being noticeably red.



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