Running the Roche Lightcycler 480

*Note: there are two profiles "carleton" and "carletonlab" on the LightCycler, which for some reason can both be accessed once we login. Login under "carletonlab". If you can't find your run or template files, look in the corresponding folder under the other profile.

Setting up a run
  1. Reserve the LightCycler using the Oracle calendar. Runs generally take 2 hours.
  2. Check and make sure the instrument and computer are turned on. If not, the power button is located on the back right of the instrument.
  3. Push the eject button on the instrument (double arrow).
  4. Make sure there are no bubbles or droplets on the plastic film that covers the plate. Place plate in instrument and close tray door by pressing the eject button again.
  5. Double click the ÒLightCycler 480Ó icon on the computer.
  6. Logon.
  7. Click "New Experiment". This will bring up a new, blank experiment file.
  8. Click "Apply template", then select "Taq Man Run Temp" (see above note if you cannot find it).
    Template consists of:
    50 deg C 2 min (1 cycle)
    95 deg C 10 min (1 cycle)
    95 deg C 10s, 55 deg C 30s, 65 deg C 1 min (35 cycles)
  9. Click "Start Run"
    *If you cannot click Start Run, check the instrument. There should be two green lights on. If there is an orange light your tray isnÕt loaded or the machine is still waking up (it can take a few minutes). There should also be a Roche LightCycler Quick Start Guide somewhere near the instrument, which has a key to what the different lights mean.
To analyze experimental data:
  1. Login as above.
  2. Click the ÒNavigator iconÓ on the right hand side of the screen (it looks like a compass).
  3. Open the Experiments folder and select the run you want to analyze.
  4. Click the "Analysis" tab.
  5. Double click the "Abs Quant / 2nd Derivative Max" tab (should be the default selection).
  6. A window entitled Create New Analysis will appear. Click the "Check icon"
  7. A window with a grid of wells, a list of samples and amplification curves will appear.
  8. Click "Calculate: at the bottom of the screen to obtain critical cycle numbers for your samples.
  9. Right click anywhere in the samples list and export a txt tab delimited file.
    *Note: in some runs toggling between High Sensitivity and High Confidence (bottom right hand tab) can give critical cycle numbers for uncertain or even negative samples. (Toggling does not change the critical cycle numbers for the rest of the samples.)
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