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

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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 requires:


-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).


Image:3AAssembly.gif
(Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly)

  • The restriction (30μL) were done as follows:


-H2O ------------> 13μl

-Buffer 2 ------> 4μl

-BSA ------------> 1μl

-DNA ------------> 10μl

-Enzyme I ----> 1μl

-Enzyme II ---> 1μl


  • 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:


1. Ladder
2. Restriction (R.) of luciferase
3. R. TT PCR
4. R. TT plasmid with XBA and PST
5. R. TT plasmid with ECO and XBA
6. R. plasmid 17
7. Positive Control of the gel (luciferase PCR Product with ECO and SPE)


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