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

  • 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 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.