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Technical Note

®

TM

Bio-Rad iCycler & iQ

Real-Time PCR Run Setup Instructions
for qBiomarker Mutation PCR Arrays
Before the Experiment
 Please make sure the real-time PCR instrument is working properly. Refer to the manufacturer’s Installation and
Maintenance manual if needed.

Creation of PCR Protocol Template
Before the Experiment (Presetting the Machine will save time for your run):
1)
2)

Open the iCycler iQ Optical System software v3.1 on the computer that is connected to the Bio-Rad iCycler
iQ Real-time PCR Detection system.
Click on the Library module on the left panel. Select the View Protocol tab. Under Protocol Files, select
2Step.tmo. Click Edit This Protocol on the right; this will open the Edit Protocol tab in the Workshop
module. Edit the thermal profile in the table at the bottom of the screen as follows (See Fig 1):
a) Click on the Dwell Time cell of the Cycle 1, Step 1 and change the value to 10:00 minutes with
Setpoint 95.0°C.
b) For Cycle 2, Step 1, change the Dwell Time value to 0:15 (15 seconds) with Setpoint 95.0°C.
For Cycle 2, Step 2, change the Dwell Time value to 1:00 minute with Setpoint 60.0°C.
Double check the value for the Repeats cell in Cycle 2 is 40.
c)
d)

Under Select data collection step(s), double check that REAL-TIME detection is selected for Cycle
2, Step 2.
Enter qBiomarker_Mutation_PCR_Array_Template as the new filename in the field for Protocol
Filename. Choose Save this Protocol to save the template.

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Technical Note

Figure 1: Protocol Setup

3)

To set up a template file for Plate Setup, click on the Library module on the left panel and then select the
View Plate Setup tab; click Create a New Plate Setup on the right. This will open the Edit Plate Setup
tab in the Workshop module. Choose the Whole Plate mode. In the Samples: Whole Plate loading tab
(See Figure 2), click to select the icon for Unknown. In the diagram of the 96-well plate, click on each well
one at a time to designate the sample type as Unknown and number the well.

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Technical Note

Figure 2: Plate Setup(1)

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Technical Note

a)

b)

When finished loading samples, click on Select and load fluorophores (See Figure 3). Select FAM
as the fluorophore by clicking the box next to the fluorophore in the Select or Deselect a Fluorophore
box. Assign a color for FAM by clicking on one of the crayons. With the FAM crayon selected, load
fluorophore to all wells by clicking the top left corner of the plate to select all wells at once. Enter
qBiomarker_Mutation_PCR_Array_Plate_Setup_Template as the new filename in the Plate Setup
Filename box.
Click Save this plate setup to save the file as a *.pts fil

Figure 3: Plate Setup (2)

Performing Real-Time PCR Detection
1)
2)
3)
4)
5)

If the thermocycler is off, turn on the iCycler and log onto the instrument. Allow the camera to warm up for
30 minutes, then switch on the computer connected to the thermocycler.
Ensure that the plate has been centrifuged for 1 min at 1000 g to remove any bubbles.
Open the thermocycler cover to expose the thermal block. Place your plate in the plate holder with the last
row (row H) facing front. Well A1 should be positioned at the top-left corner of the holder. Close the cover.
Open the iCycler iQ Optical System software.
In the Library module, click to open the View Protocol tab.
a) Under Protocol Files, click to highlight and load the previously saved
qBiomarker_Mutation_PCR_Array_Template file.
b) Click to open the View Plate Setup tab; under Plate Setup Files, click to highlight and load the
qBiomarker_Mutation_PCR_Array_Plate_Setup_Template file.

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Technical Note

c)

6)

Click Run with Selected protocol to initiate the run with the selected plate setup and indicated
protocol.
d) This will lead you to the Run Prep tab in the Workshop module.
In the Run Prep tab:
a) Enter the reaction volume (25 µL) for the Sample volume.
b) Select Well Factor Plate under Select Well Factor Source.

If using the iCyler iQ, iQ5, or MyiQ, well factors must be collected at the beginning of each
experiment. Well factors are used to compensate for any system or pipetting nonuniformity.
c) When ready, click on Begin Run. A Save dialog box will open. Enter a name for the optical data file
(*.opd).

After the PCR Run
1)

2)
3)

4)

5)

Select the Library module and click on the View Post-Run Data tab. Select a data file and click Analyze
Data on the right under Data Analysis Operations. The data file will open in the PCR Quantification tab
of the Data Analysis module. Select PCR Base Line Subtracted Curve Fit in the field for Select
analysis mode.
Choose FAM in the Select a Reporter box.
To determine CT values, Set Baseline at 3 to 18 cycles. To define the Threshold value, use the Log View
of the amplification plot and place it above the background signal but within the lower half to one-third of the
linear phase of the amplification plot.
Click on the Reports button to obtain the customized reports of the PCR quantification data, the window for
Report Viewer will open. On the left panel under Select Report, select to view Melt and Amp Data. To
save the report, select Save to File, and enter a filename for the report to be saved in the Rich Text Files
format.
Close the Report Viewer window, and check the Open/Save Settings and select Save OPD File to save
the data file with the current analysis parameters.

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