User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/05

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Ligation and Transformation of luciferase and double terminator
    1. Ladder 2. Blank 3. Double terminator | BBa_B0015 4. Restriction of luciferase 5. Negative control (water) 6. Positive control (plasmid pSB1C3 with luciferase but without restriction)    1. Description  <br/ > <br/ > -Plasmid pSB1C3 represented in the image as the red construction and restricted with ECO RI and PST I.<br/ > -The luciferase represented as part 1 and restricted with ECO RI and SPE I. <br/ > -The double terminator (TT) represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I). <br/ > <br/ > <br/ > <br/ > <br/ > (Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly) <br/ > <br/ > 2. Restrictions: <br/ > <br/ > <br/ > -H2O > 13μl<br/ > -Buffer 2 ---> 4μl<br/ > -BSA > 1μl<br/ > -DNA > 10μl<br/ > -Enzyme I --> 1μl<br/ > -Enzyme II -> 1μl<br/ > <br/ > <br/ > <br/ > <br/ > <br/ > 1. Ladder.<br/ > 2. Restriction (R.) of luciferase.<br/ > 3. R. TT PCR.<br/ > 4. R. TT plasmid with XBA I and PST I.<br/ > 5. R. TT plasmid with ECO RI and XBA I.<br/ > 6. R. plasmid pSB1C3.<br/ > 7. Positive Control of the gel (luciferase PCR Product with ECO RI and SPEI).<br/ > <br/ > <br/ > 3. Ligations <br/ > <br/ > <br/ > 1: luciferase plasmid + TT PCR <br/ > 2: luciferase plasmid + TT plasmid <br/ > 3: luciferase PCR + TT PCR <br/ > 4: luciferase PCR + TT plasmid <br/ > -H2O > 7μL<br/ > -Buffer ligase --> 2μL<br/ > -Vector (plasmid) -> 2μL<br/ > -Insert (luciferase) ---> 3μL<br/ > -Insert (double ter.) -> 5μL<br/ > -Ligase -> 1μL<br/ > <br/ > <br/ > 4. Transformation <br/ > <br/ > <br/ >
 * The first thing to do was to quantify the vector-insert relation for the ligation, I ran a 2% agarose gel for 50 min at 90V:
 * The lanes are as follow:
 * Three antibiotic Assembly
 * As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a | Three Antibiotic Assembly. As 3 parts are required:
 * The restriction (30μL) were done as follows:
 * I left the restrictions incubating for 2hrs at 37°C and inactivated the enzymes at 65°C for 10 min. After this, I ran a gel at 90V for 1hr.
 * The lanes are:
 * I did four with different combinations to optimize the probabilities of getting colonies transformed with the ligation.
 * The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT.
 * Reaction Mix
 * I transformed competent cells and plated them in tetracycline-LB plates. They grew well, so I replated them and extracted plasmid by using the Roche High Pure Plasmid Isolation Kit.
 * Finally, I did a restriction for a total of 20μL using ECO RI and PST I, and I left them incubating at 37°C ON(overnight). The gel can be checked here.

Proving that the ligation was done correctly
<br/ >
 * As a way to check that now I have my device complete (luciferase + double terminator), we synthesize a special primer containing a


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