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

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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