User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/07

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Ligation day!!!


  • As in the previous days there was no success in the ligations, I did four with different combinations in order to optimize the probabilities of getting colonies transformed with both, luciferase and the double terminator.


  • 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. All ligations were labeled as lig. 1-4 TAA M, ligación lucif + TT Mar


  • 1: luciferase plasmid + TT PCR (1-F)


-H2O -----------------------> 6μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 3μL
-Insert (luciferase) ---> 3μL
-Insert (double ter.) --> 5μL
-Ligase --------------------> 1μL

  • 2: luciferase plasmid + TT plasmid


-H2O -----------------------> 7μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 3μL
-Insert (double ter.) --> 5μL
-Ligase --------------------> 1μL

  • 3: luciferase PCR + TT PCR


-H2O -----------------------> 7μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 3μL
-Insert (double ter.) --> 5μL
-Ligase --------------------> 1μL

  • 4: luciferase PCR + TT plasmid


-H2O -----------------------> 7μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 3μL
-Insert (double ter.) --> 5μL
-Ligase --------------------> 1μL


  • After the ligation, I transformed competent cells and plated them in tetracycline-LB plates.