User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/29

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When work takes over...


  • As soon as I arrived to the lab, I checked that my strains were growing well. I inoculated them in 4ml of LB with their respective antibiotic for ~7hr.


  • Next, I extracted plasmid with the High Pure Plasmid Isolation Kit of Roche. Products were stored as p17/J23100/lumP(1-4) Mar Plasm Pur and the date respectively.


  • Finally, I did the following restrictions so that tomorrow I am ready to ligate.


1. lumP with XBAI and PST I stored as lumP 1 R-> XBA PST Mar and the date.

-H2O -------> 8μl
-Buffer 3 --> 3μl
-BSA ------> 1μl
-DNA -------> 15μl
-XBA I -----> 1.5μl
-PST I ------> 1.5μl

2. lumP with XBA I stored as lumP 1 R-> XBA Mar and the date.
-H2O -------> 11μl
-Buffer 4 --> 2μl
-BSA ------> 1μl
-DNA -------> 5μl
-XBA I -----> 1μl

3. lumP with PST I stored as lumP 1 R-> PST Mar and the date.
-H2O -------> 11μl
-Buffer 3 --> 2μl
-BSA ------> 1μl
-DNA -------> 5μl
-PST I -----> 1μl

4. Green luciferase with ECO RI and PST I stored as luc wt R-> ECO y PST Mar and the date.

-H2O -------> 13μl
-Buffer 3 --> 3μl
-BSA ------> 1μl
-DNA -------> 10μl
-ECO I -----> 1.5μl
-PST I ------> 1.5μl

5. Mutated-luciferase with ECO RI and PST I stored as mut-luz R-> ECO y PST Mar and the date.
-H2O -------> 13μl
-Buffer 3 --> 3μl
-BSA ------> 1μl
-DNA -------> 10μl
-ECO RI -----> 1.5μl
-PST I -------> 1.5μl

6. Plasmid PSB1C3 with each enzyme where ECO RI (buffer 2), XBA I (buffer 4), SPE I (buffer 4), and PST I (buffer 3) stored as p17 R-> enzyme Mar and the date.
-H2O -------> 11μl
-Buffer -----> 2μl
-BSA ------> 1μl
-DNA -------> 5μl
-Enzyme ----> 1μl

7. J23100 with SPE I and PST I stored as J23100 R-> SPE y PST Mar and the date.
-H2O -------> 8μl
-Buffer 2 --> 3μl
-BSA ------> 1μl
-DNA -------> 15μl
-SPE I ----> 1.5μl
-PST I ----> 1.5μl

8. J23100 with each enzyme where ECO RI (buffer 2), XBA I (buffer 4), SPE I (buffer 4), and PST I (buffer 3).
-H2O -------> 11μl
-Buffer -----> 2μl
-BSA ------> 1μl
-DNA -------> 5μl
-Enzyme ----> 1μl

  • Restrictions were incubated at 37°C ON.