User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/05: Difference between revisions
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** '''Three antibiotic Assembly''' | ** '''Three antibiotic Assembly''' | ||
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1. Description | |||
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* As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly| Three Antibiotic Assembly]. As 3 parts are requires: | * As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly| Three Antibiotic Assembly]. As 3 parts are requires: | ||
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2. Restrictions: | |||
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* The restriction (30μL) were done as follows: | * The restriction (30μL) were done as follows: | ||
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-H<sub>2</sub>O ------------> 13μl<br/ > | -H<sub>2</sub>O ------------> 13μl<br/ > | ||
-Buffer 2 ------> 4μl<br/ > | -Buffer 2 ------> 4μl<br/ > | ||
-BSA ------------> 1μl<br/ > | -BSA ------------> 1μl<br/ > | ||
-DNA ------------> 10μl<br/ > | -DNA ------------> 10μl<br/ > | ||
-Enzyme I ----> 1μl<br/ > | -Enzyme I ----> 1μl<br/ > | ||
-Enzyme II ---> 1μl<br/ > | -Enzyme II ---> 1μl<br/ > | ||
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3. Ligations | |||
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* The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT. | * The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT. | ||
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1: luciferase plasmid + TT PCR | |||
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2: luciferase plasmid + TT plasmid | |||
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3: luciferase PCR + TT PCR | |||
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4: luciferase PCR + TT plasmid | |||
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* '''Reaction Mix''' | * '''Reaction Mix''' | ||
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-Ligase --------------------> 1μL<br/ > | -Ligase --------------------> 1μL<br/ > | ||
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4. Transformation | |||
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* I transformed competent cells and plated them in tetracycline-LB plates. They grew well, so I replated them and extracted plasmid by using the Roche High Pure Plasmid Isolation Kit. | |||
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* Finally, I did a restriction for a total of 20μL using ECO RI and PST I, and I left them incubating at 37°C ON(overnight). The [[User:Mariana_Ruiz_Velasco_L./Notebook/IGEM_2010/Wet_lab_journal/2010/06/10| gel can be checked here]]. | |||
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Revision as of 19:39, 13 August 2010
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Ligation and Transformation of luciferase and double terminator
-H2O -----------------------> 7μL
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