User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/25

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Luciferase ++


  • Early in the morning, I ran a 1% low fusion point agarose gel to make a band gel purification. The gel ran 73 min at 85V and the band was cut after dying the gel. The band weighted 380μg.


  • After the purification, I made 3 restrictions (30μL) so that ligations can take place next:


1. Plasmid with constitutive promoter stored as J23106 c/SPE y PST Mar (1-N).

-H2O -------> 8μl

-Buffer 2 --> 3μl

-BSA ------> 1μl

-DNA -------> 15μl

-SPE I -----> 1.5μl

-PST I ------> 1.5μl

2. Plasmid pSB1T3 a.k.a. 18 stored as P 18 c/XBA y PST Mar (1-O).

-H2O -------> 8μl

-Buffer 2 --> 3μl

-BSA ------> 1μl

-DNA -------> 15μl

-XBA I -----> 1.5μl

-PST I ------> 1.5μl

3. Mutated luciferase purified from band (4-M) and stored as Mut-luz c/XBA y PST Mar (1-P).

-H2O -------> 8μl

-Buffer 2 --> 3μl

-BSA ------> 1μl

-DNA -------> 15μl

-XBA I -----> 1.5μl

-PST I ------> 1.5μl

Dye Another Gel


  • To check and quantify the restrictions (if the product is as expected) I ran a 0.8% agarose gel for 50min at 90V.




  • The lanes are as follows:


1. Ladder.

2. Restriction of J23106.

3. Restriction of Plasmid 18.

4. Restriction of Mutated luciferase.

5. Purified Mutated luciferase from gel.

Ligating


  • The final thing to do today (after dephosphating 20 min at 37°C and inactivating 10 min at 65°C both plasmids. Labeled as J23106 and P18 defosfat. Mar 25/06 respectively). This was left ON at 37°C.


  • 1: luciferase (green) with J23106


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

  • 2: luciferase (green) with plasmid 18


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

  • 3: luciferase (red) with J23106


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

  • 4: luciferase (red) with plasmid 18


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

  • 5: Control (J23106)


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

  • 6: Control (plasmid 18)


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


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