User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/04

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 * style="background-color: #EEE"|[[Image:Logo_light_minimal_full_res.jpg|150px]] WiFi coli: A communicolight system
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Three antibiotic Assembly
  -The plasmid 17 (pSB1C3) represented in the image as the red construction and restricted with ECO RI and PST I. -The luciferase represented as part 1 and restricted with ECO RI and SPE I.  -The double terminator 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).   
 * 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 requires:

 (Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly)    -H2O > 13μl
 * The restriction (30μL) were done as follows:

-Buffer 2 --> 4μl

-BSA > 1μl

-DNA > 10μl<br/ >

-Enzyme I > 1μl<br/ >

-Enzyme II ---> 1μl<br/ > <br/ > <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 and PST<br/ > 5. R. TT plasmid with ECO and XBA<br/ > 6. R. plasmid 17<br/ > 7. Positive Control of the gel (luciferase PCR Product with ECO and SPE)<br/ > <br/ >
 * 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:


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