User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/09/21

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Working harder than ever with luciferases...

  • Today, I inoculated luciferases from photinus pyralis (with mutated RBS) of strains 3, 5, 8 and its mutated version (strains 6,8,9) in 4ml of LB with 4μl of ampicilin and leave them growing at 37°C for 6 hours.

  • After extracting plasmid with the High Pure Plasmid Isolation Kit from Roche®, I did a restriction for a total of 30μL as follows to check if the luciferase was ligated in the pBSIIK(+/-):

-H2O -------> 13μl

-Buffer 4 --> 3μl

-BSA -------> 1μl

-DNA ------> 10μl

-ECOR1 -----> 1.5μl

-PST1 ------> 1.5μl

  • Restrictions were incubated at 37°C ON (overnight) and labeled as luc or mluc No. R-> ECO y PST Mar. and date.

  • Melvin is also helping with the experiment, he did the same as me but for the Promega® click beetle luciferases, cutting them with XBAI and PSTI.

  • I also inoculated 3 strains containing luciferase from photinus pyralis in 4ml of LB with 4μl of kanamycin and leave them growing at 37°C ON.