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

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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    -H2O -> 13.5μl <br/ > -Buffer ---> 6μl <br/ > -Mg(OAc) > 3μl <br/ > -dNTPs(0.4mM c/u) > 2μl <br/ > -oligo FWD (prefix, 5pmol/μL) ---> 2μl <br/ > -oligo RVS (suffix, 5pmol/μL) > 2μl <br/ > -DNA -> 1μl <br/ > -rtTh(high fidelity)-> 0.5μl <br/ > <br/ > <br/ > -positive: plasmid 18<br/ > -negative: blank <br/ > <br/ > <br/ > - Denaturation step: 94°C for 45 sec.<br/ >
 * 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:<br/ >
 * The controls were:
 * We program the thermo-cycler as follows for 30 cycles:

- Annealing step: 60°C for 45 sec. <br/ >

- Extension/elongation step: 72°C for 2:00 min.<br/ >

- Final elongation: 72°C for 10:00 min.<br/ >

- Final hold: 4°C for ∞. <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > -H2O --> 11μL<br/ > -Buffer ---> 3μL<br/ > -DNA -> 15μL<br/ > -Phosphatase --> 1μL<br/ > <br/ > <br/ >
 * 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:
 * The reaction was incubated at 37°C for 20 min and at 65°C for 10min.


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