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

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Luciferase ++
   1. Plasmid with constitutive promoter stored as J23106 c/SPE y PST Mar (1-N).  -H2O ---> 8μl
 * 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:

-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<br/ >

-Buffer 2 --> 3μl<br/ >

-BSA --> 1μl<br/ >

-DNA ---> 15μl<br/ >

-XBA I -> 1.5μl<br/ >

-PST I --> 1.5μl<br/ > <br/ > 3. Mutated luciferase purified from band (4-M) and stored as Mut-luz c/XBA y PST Mar (1-P).<br/ > <br/ > -H2O ---> 8μl<br/ >

-Buffer 2 --> 3μl<br/ >

-BSA --> 1μl<br/ >

-DNA ---> 15μl<br/ >

-XBA I -> 1.5μl<br/ >

-PST I --> 1.5μl<br/ > <br/ >

Dye Another Gel
<br/ > <br/ > <br/ > <br/ > <br/ > 1. Ladder.<br/ >
 * To check and quantify the restrictions (if the product is as expected) I ran a 0.8% agarose gel for 50min at 90V.<br/ >
 * The lanes are as follows:

2. Restriction of J23106.<br/ >

3. Restriction of Plasmid 18. <br/ >

4. Restriction of Mutated luciferase. <br/ >

5. Purified Mutated luciferase from gel. <br/ > <br/ >

Ligating
<br/ > <br/ > <br/ > -H2O ---> 9μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 5μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 9μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 5μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 8μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 6μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 8μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 6μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 14μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 14μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Ligase > 1μL<br/ > <br/ >
 * 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
 * 2: luciferase (green) with plasmid 18
 * 3: luciferase (red) with J23106
 * 4: luciferase (red) with plasmid 18
 * 5: Control (J23106)
 * 6: Control (plasmid 18)


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