User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/30

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Let's rethink the methods :(

  • As my gel showed only 2 bands, we decided that it would be better to extract plasmid directly from a culture. The strains I grew were inoculated in 4ml of LB with 4μl of their respective resistance for approximately 5 hours.

  • The next step was to extract and purify the plasmid using the Roche's high plasmid purification kit. As strains of the plate 1 (green luciferase + J23106 promoter) didn't grow well, I left them and did the following ones:

2.2, 2.8, 3.1, 3.3, 3.5, 3.8, 3.9, 4.2, 4.3, 4.7, 4.8, and 4.9 (all of them labeled as Mar Plasm Purif and the date).

  • Finally, I did a restriction for a total of 20μL as follows:

-H2O -------> 10μl

-Buffer 4 --> 2μl

-BSA -------> 1μl

-DNA ------> 5μl

-ECOR1 -----> 1μl

-PST1 ------> 1μl

  • Restrictions were incubated at 37°C ON(overnight) and labeled as their code ECO PST and the date.

NOTE: All tubes were stored in the fridge at 4°C.