User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/06

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  • I'm ready to do the restrictions and ligations (FINALLY!!!) for the pBSKII (+/-) vector so that I can finally prove the mutation. At the same time, I will be using pSB1C3, as it is the standard for iGEM's 2010 DNA submission. The restrictions:


1. Mutated luciferase with ECO RI and PST I stored as mut-luz R-> ECO y PST Mar and the date.

-H2O --------> 12μl
-Buffer 3 --> 3μl
-BSA -------> 1μl
-DNA -------> 10μl
-ECO I -----> 2μl
-PST I ------> 2μl

2. pSB1C3 with ECO RI and PST I stored as p17 R-> ECO y PST Mar and the date.

-H2O --------> 12μl
-Buffer 3 --> 3μl
-BSA --------> 1μl
-DNA --------> 10μl
-ECO RI ----> 2μl
-PST I -------> 2μl

  • The gel:



Restr 6Ago10.JPG

  • The lanes:


1,7. Ladder.

2. Mutated luciferase restricted with ECO RI and PST I.

3. p17 restricted with ECO RI and PST I.

4,5,6. Things from Zepeda and Augusto.


Ligations... again :D


  • 1: luciferase (green) with pBSKII


-H2O -----------------------> 9μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 6μL
-Ligase --------------------> 1μL

  • 2: luciferase (green) with plasmid 17


-H2O -----------------------> 8μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 3μL
-Insert (luciferase) ---> 6μL
-Ligase --------------------> 1μL

  • 3: luciferase (red) with pBSKII


-H2O -----------------------> 10μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 5μL
-Ligase --------------------> 1μL

  • 4: luciferase (red) with plasmid 17


-H2O -----------------------> 9μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 3μL
-Insert (luciferase) ---> 5μL
-Ligase --------------------> 1μL

  • 5: Control (pBSKII)


-H2O -----------------------> 15μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 2μL
-Ligase --------------------> 1μL

  • 6: Control (plasmid 17)


-H2O -----------------------> 14μL
-Buffer ligase -----------> 2μL
-Vector (plasmid) -----> 3μL
-Ligase --------------------> 1μL

  • The reactions were left at 4°C during the whole weekend.