User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/16

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Getting ready for punctual mutation

  • Today, I inoculated E. coli strains containing a plasmid with a constitutive promoter so that, after 6-7 hrs of incubation at 37°C, I can extract the plasmid and ligate the luciferase and double terminator with it.

  • I also did a restriction with Xba I and Pst I for a total of 30μL as follows:

-H2O -------> 18μl

-Buffer 2 --> 4μl

-BSA ------> 1μl

-DNA -------> 5μl

-XBA I -----> 1μl

-PST I ------> 1μl

  • Restrictions were incubated at 37°C for 4 hr and labeled as: lucif + TT c/rbs + 1 purif restr. XBA y PST Mar and the date in the top of the eppendorf.

  • I also ran a gel at 90V for 50min to quantify the ligation PCR product with the mutated RBS to know how to prepare the mix for the PCR with Pfx.
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