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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* 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 | 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 required: | |||
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-Plasmid [http://partsregistry.org/Part:pSB1C3 pSB1C3] represented in the image as the red construction and restricted with ECO RI and PST I.<br/ > | -Plasmid [http://partsregistry.org/Part:pSB1C3 pSB1C3] represented in the image as the red construction and restricted with ECO RI and PST I.<br/ > | ||
-The luciferase represented as part 1 and restricted with ECO RI and SPE I. <br/ > | -The luciferase represented as part 1 and restricted with ECO RI and SPE I. <br/ > | ||
-The double terminator represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I). <br/ > | -The double terminator (TT) represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I). <br/ > | ||
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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''' | ||
-H<sub>2</sub>O | -H<sub>2</sub>O --------------------> 7μL<br/ > | ||
-Buffer ligase | -Buffer ligase ----------> 2μL<br/ > | ||
-Vector (plasmid) -----> 2μL<br/ > | -Vector (plasmid) -----> 2μL<br/ > | ||
-Insert (luciferase) ---> 3μL<br/ > | -Insert (luciferase) ---> 3μL<br/ > | ||
-Insert (double ter.) | -Insert (double ter.) -> 5μL<br/ > | ||
-Ligase | -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]]. | |||
==Proving that the ligation was done correctly== | |||
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* As a way to check that now I have my device complete (luciferase + double terminator), we synthesize a special primer containing a | |||
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Revision as of 12:04, 26 September 2010
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Ligation and Transformation of luciferase and double terminator
-H2O --------------------> 7μL
Proving that the ligation was done correctly
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