User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/04/27

WiFi coli: A communicolight system

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Experimental procedure to obtain luciferase and double terminator We decided to obtain the luciferase by PCR, so that we could be certain of the product. Amplifying luciferase by PCR To obtain the luciferase from the plasmid (that had already been purified with Roche's Kit "High Pure Plasmid"), I realized a double restriction using ECO RI and SPEI with the following reaction for a total of 40μl: -H2O ------> 6μl -BSA ------> 1μl -Buffer ----> 4μl -ECO RI --> 2μl -SPE I ----> 2μl -DNA ------> 25μl The reaction was incubated at 37°C for 6.5 hrs followed by a 2% agarose gel electrophoresis ran for 1 hr at 85V. Miguel helped me to do the PCR in order to amplify the luciferase. The reaction was done as follows for a total of 30μl: -H2O ---------------------------------> 13.5μl -Buffer -------------------------------> 6μl -Mg(OAc) ----------------------------> 3μl -dNTPs(0.4mM c/u) ----------------> 2μl -oligo FWD (prefix, 5pmol/μL) ---> 2μl -oligo RVS (suffix, 5pmol/μL) ----> 2μl -DNA ---------------------------------> 1μl -rtTh(high fidelity)-----------------> 0.5μl The controls were: -positive: plasmid 18 -negative: blank We program the thermo-cycler as follows for 30 cycles: - Denaturation step: 94°C for 45 sec. - Annealing step: 60°C for 45 sec. - Extension/elongation step: 72°C for 2:00 min. - Final elongation: 72°C for 10:00 min. - Final hold: 4°C for ∞. The PCR product was purified and observed in the following gel: The last step before ligation was to do a restriction of the PCR purified product with ECO RI and SPEI for a total of 40μl. Double terminator I also did a restriction assay with ECO RI and XBA I of the double terminator BBa_B0015 (the one augusto had worked in), and left it at 37°C ON. Afterwards, I dephosphated it with the following reaction for 30μL: -H2O --------------> 11μL -Buffer -----------> 3μL -DNA -------------> 15μL -Phosphatase --> 1μL The reaction was incubated at 37°C for 20 min and at 65°C for 10min.