User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/09/08

WiFi coli: A communi-colight system

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It's been a hard day's night, and I'll be working like a dog I started the day by extracting plasmid from the LRE + pSB1C3 strains so that I could check if the transformation was done correctly. After making a restriction for 20μL as follows, I ran a gel for 50min at 90V. - H2O -------------> 10 - Buffer 3 ------> 2μL - BSA ------------> 1μL - DNA ------------> 5μL - ECO RI --------> 1μL - PST I ----------> 1μL The lanes were as follows and it can be observed that LRE from strains 3 and 5 was transformed successfully. 1. Ladder. 2,3,4. LRE + pSB1C3. 5. Green click beetle luciferase from purified PCR product. 6. Red click beetle luciferase from purified PCR product. 7. Mutated Photinus pyralis luciferase from purified PCR product. About the click beetle experiment, I put a restriction for a total of 30μL so that I can be able to ligate it into a plasmid containing a constitutive promoter. The reaction was as follows and it was left incubating at 37°C ON: - H2O -------------> 13 - Buffer 3 ------> 3μL - BSA ------------> 1μL - DNA ------------> 10μL - XBA I ---------> 1.5μL - PST I ----------> 1.5μL Finally, for the luciferase + plasmid with T7 promoter, I inoculated 3 strains of it and 1 of the wildtype luciferase. Two out of the 3 pT7 strains were transformed and left incubating ON at 37°C.