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''' | ||
# 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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[[Image:3AAssembly.gif]] | [[Image:3AAssembly.gif]] | ||
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(Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly) | (Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly) | ||
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# Restrictions: | |||
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* The restriction (30μL) were done as follows: | * The restriction (30μL) were done as follows: | ||
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* The lanes are: | * The lanes are: | ||
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1. Ladder<br/ > | 1. Ladder.<br/ > | ||
2. Restriction (R.) of luciferase<br/ > | 2. Restriction (R.) of luciferase.<br/ > | ||
3. R. TT PCR<br/ > | 3. R. TT PCR.<br/ > | ||
4. R. TT plasmid with XBA and PST<br/ > | 4. R. TT plasmid with XBA I and PST I.<br/ > | ||
5. R. TT plasmid with ECO and XBA<br/ > | 5. R. TT plasmid with ECO RI and XBA I.<br/ > | ||
6. R. plasmid pSB1C3<br/ > | 6. R. plasmid pSB1C3.<br/ > | ||
7. Positive Control of the gel (luciferase PCR Product with ECO and | 7. Positive Control of the gel (luciferase PCR Product with ECO RI and SPEI).<br/ > | ||
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# Ligations | |||
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* I did four with different combinations to optimize the probabilities of getting colonies transformed with the ligation. | |||
* 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''' | |||
-H<sub>2</sub>O -----------------------> 7μL<br/ > | |||
-Buffer ligase -----------> 2μL<br/ > | |||
-Vector (plasmid) -----> 2μL<br/ > | |||
-Insert (luciferase) ---> 3μL<br/ > | |||
-Insert (double ter.) --> 5μL<br/ > | |||
-Ligase --------------------> 1μL<br/ > | |||
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* After the ligation, I transformed competent cells and plated them in tetracycline-LB plates. | |||
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Revision as of 19:30, 13 August 2010
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Ligation and Transformation of luciferase and double terminator
-Buffer 2 ------> 4μl -BSA ------------> 1μl -DNA ------------> 10μl -Enzyme I ----> 1μl -Enzyme II ---> 1μl
-H2O -----------------------> 7μL
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