Imperial College/Courses/Spring2008/Synthetic Biology/Cellular And Molecular Biology Practical/Day 2

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 Wet Lab: EcoRV Restriction Endonuclease over expression and digestion of DNA

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Schedule

8.30 am
 * 1) Inoculate TWO 25 ml cultures from your overnight cultures by aseptically adding 1ml of overnight culture to the autoclaved flask. Add an appropriate volume of ampicillin antibiotic to provide the same concentration that was used in the overnight cultures. Place in the 28°C incubator.
 * 2) Place your other two flasks in the 55°C water bath.
 * 3) Update your lab notebook.

12.00
 * 1) Remove your flasks from the 28°C, take 1ml of each culture and place in a cuvette.
 * 2) Remove flasks from the 55°C water bath and add ampicillin. Take 1 ml of this fresh media into a single cuvette (to be used as a blank). WORK QUICKLY.
 * 3) Add one flask of hot media to each flask growing at 28°C and QUICKLY return to the incubator. Make sure the incubator temperature is increased to 42°C.
 * 4) You should now have 3 cuvettes, one with clean media and two with your bacterial cultures. Use the fresh media to zero the spectrophotometer at 550 nm. Now read the OD550 of each bacterial culture.(Question 2: At what OD have you induced your cells?)
 * 5) Update your lab notebook.

4.00pm
 * 1) Remove flasks from incubator and pour cultures into 50 ml centrifuge tubes.
 * 2) Spin down in centrifuge for 15 mins. (make sure the rotor is balanced; work with others to fill up the centrifuge).
 * 3) Pour off supernatant back into 250 ml flasks. [Safety note: spent media must not be poured down the sink, bacterial waste must be sterilised before it leaves the building. These flasks will be autoclaved before the media is discarded.]
 * 4) Label your tubes and place in the racks provided to be stored in the freezer.
 * 5) Update your lab notebook.