Procedure for Reactions Done with the Ocean Optics Instrument
Lab bench setup:
- Fill an ~8 Qt container with ice, then add water until mostly full
- Place pump portion of the Ocean Optics & the end of the yellow tube in the ice bath
- Note that there are two sections of yellow tubing. One section should be connected to the pump and the Ocean Optics machine. The other should have one end connected to the Ocean Optics machine, while the other is allowed to rest/drain back into the ice bath.
When making the solutions for the Ocean Optics:
- 15mg lyoph. protein/1ml solvent
- 0.114ml 30% peroxide/0.887ml solvent
- 1mg OPD/1ml solvent
Computer and Instrument setup:
- Turn on the Jaz apparatus of the Ocean Optics instrument (simply press the power button, no other buttons need to be pressed)
- Turn on the light source apparatus of the Ocean Optics instrument (switch on the back of the machine), then turn on the Deuterium lamp (blue button). Allow the Deuterium lamp to be on for 30 minutes (or until finished flashing) before you begin the experiment, and before you turn on the Halogen lamp.
- Turn on the Halogen lamp.
- Open the Q-Blue Wireless Temperature Control software from the desktop.
- Check the box for control status (it should read "on"), change the target temperature to 25 degrees C, and make sure the stirrer is on and set to 1000 rpm.
- Open the OceanView software from the desktop.
- Run a wizard, select Spectoscopy, select Jaz as the data source, and select absorbance.
- Set integration time to 1 ms, change to 10 scans to average
- Click big yellow light bulb to get a light spectrum (should look like a normal, linear, not crazy graph) and hit next. Flip the switch on the light source toward the right to turn the light off, then click the big grey light bulb to get a dark spectrum and hit next. Turn the light back on.
- You should now see the graph. Click on the paper+wrench icon (to the left of the print icon).
- Click yes to popup. Change it to 1 minute between every scan, and make it stop after 2 hours. Set it to 4 padding digits. Change the basename to something like: Mb_KCl_Tris_OPD_1_Propanol_25C. Change the save location to a folder for the day created within Documents>OceanView Ocean Optics>2014>month. Hit apply and close window (note that the window will not automatically close after you select apply).
When putting solutions into cuvette:
- Put 2.025ml (0.675 * 3) of OPD solution and 0.75ml peroxide solution into the cuvette in the Ocean Optics Temperature Control apparatus.
- One after another on the computer, click the save icon, click the play button, and then click the save icon again to begin data collection.
- Check to make sure your data is being saved properly by looking at the folder you created in Documents>OceanView Ocean Optics>2014>month.
- Before third keep, put 0.225ml of the protein solution in the cuvette.
When data collection is finished:"
- Exit out of the graph window. Completely turn off the light source apparatus and the Jaz apparatus. Unplug the pump and the Temperature Control apparatuses. Remove the pump from the ice bath, set it on the lid of the bucket, and pour the ice bath into the sink. Turn off the computer and monitor(s).
- After all observations of the solution are made and recorded, pour the solution into an appropriate waste container (being extremely mindful not to lose the tiny stir bar in the cuvette). Remove the stir bar from the cuvette, and wash the cuvette and stir bar separately (to prevent the stir bar from being lost down the drain of the sink) with deionized water three times. Dry the cuvette and stir bar with a Kimwipe, place the stir bar back into the cuvette, and place the cuvette in the cuvette tray.