Press the eject button and wait until the drawer completely opens.
Make sure notch on plate is in the bottom right corner.
Place plate in drawer and push drawer until it completely closes.
How to start a new experiment
Click on the “New” icon on the right hand side of the screen
Choose from the three options given:
New empty experiment
New experiment based on existing experiment
New experiment based on Roche template
For a “New empty experiment”: once selected update the “Experiment name”, click ok, then create.
Under the “Measurement” tab (found on top left), click on the pencil icon in the center of the screen to select the channel of choice and the dye of choice within the channel. Click back to return.
Update the reaction volume and press ok.
Click on the “Profile” tab (next to the measurement) to add program steps.
Click on the plus icon on the left hand side and chose from the following six options:
Preincubation
3 step amplification
High resolution melting
2 step amplification
Melting
Cooling
Highlight the step you want to add by clicking on it and press “add” on the right side. (Do this for all steps desired)
Click back
Update each steps hold time and temperature target by selecting the pencil icon in the center of the screen (to the left of the “Step” column)
Update each steps cycle number by selecting the step on the left and selecting the pencil icon on the far left (to the left of the “Program” column)
For the annealing step there are three options available:
Standard: every well will have same temperature
Gradient: allows you to select a temperature range between columns 1-12
Click “details” to see the specific temperature on each well
Touchdown: allows you to start the first cycle at a higher temperature and have the temperature change at each step ending at the target temperature
Once all parameters have been updated, click on the “Eject” button on the far right to open the drawer.
Place plate inside drawer and close drawer
Once the drawer is closed, start experiment by clicking on the “Start” icon on the far right
How to start a new experiment based on an existing experiment
Click on the “New” icon on the right hand side of the screen
Select “New experiment based on existing experiment”
Click on the pencil icon on the right to select from the list of existing experiments.
Select the experiment you would like to clone and click ok
Update the “Experiment name”, click ok, then create.
How to start a new experiment based on an existing Roche template
Click on the “New” icon on the right hand side of the screen
Select “New experiment based on Roche template”
Click on the pencil icon on the right to select from the list of Roche experiments.
Select the experiment you would like to clone and click ok
Update the “Experiment name”, click ok, then create.
Visualize Raw Data
Select “Raw data” tab on the top to view fluorescence over time
You can switch between channels by clicking on the tabs under the graph
The left and right arrows above the graph will display a fluorescence heat map
Labeling Samples
Can view samples as plate view or table view (top right tabs)
Clearing wells is very important when you do not have samples in them so they are not included in the background calculation
Select wells that are empty and click “Clear wells” on the bottom right
To label samples, highlight the wells relevant to a given gene and enter the gene name in the box on the right
Individually enter name and sample type, or if triplicate, for example, highlight all three and enter the sample name and type on the right
If you have a set of standards: highlight all of the standard curve wells, name them, chose “standard” sample type and chose the “auto standard curve” icon to generate concentrations – be sure to select if the series starts with the highest or lowest concentration in the window that appears.
Absolute Quantification
Click on “Analysis” tab
Click on the plus icon (above the analysis tab on the far right)
Select Abs Quant option and click ok
Software will calculate the standard curve slope, efficiency, error, R2, and Y-intercept
RelQuant
If not already done - Open experimental file on lightcycler 96 user software
Enter the sample information in the sample editor
Click on “Analysis” tab
Click on the plus icon (above the analysis tab on the far right)
Select Rel Quant option and click ok
Select the reference gene in the pop-up window by checking the box in the “reference column” under the correct gene
Under the “sample” tab, indicate which sample you will use as the run calibrator – check the box under the “Run calibrator” column
Under the “Conditions” tab, can chose the conditions you want to compare the others to (for example time 0)