IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/21

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July 21, 2010
With the cloned out parts and parts from the registry, we began testing digestion/ligation/transformation.

Each one was digested with the following procedure. The restriction enzymes and volume of template of each is on the table above:
 * RXN (50 uL)
 * 5 uL NEBuffer 2
 * 0.5 uL BSA
 * 1.0 uL Restriction Enzyme 1
 * 1.0 uL Restriction Enzyme 2
 * Volume of template from table
 * Volume of dH20 that will make the reaction 50 uL overall

After all of the restriction enzyme and other solutions were added to the reactions, they were put in a 37 C bath for 30 minutes. After that, they were put in a 80 C bath for 20 minutes.

Next ligations of all the digestions was done with the following procedure:

Each was incubated for 15 minutes at room temperature (25 C)

Then they were transformed into 50 uL of dH5α cells with 3 uL of ligation solution.

Ligation 1 was inoculated into 200 uL of LB/Kanamycin broth and overnighted in the 37 C room. All the rest of the ligations were inoculated into 200 uL of LB/Chloramphenicol broth and put in the 37 C overnight.