Cycling The Calibration Plate; Image Optimization - BIO RAD iCycler iQ Packing List And Installation Manual

2. In a 10–15 ml plastic disposable polypropylene tube, prepare a dilution of the
400 nM solution:
Dilution 3 300 µl, 400 nM (Dil 2)
3. Pipette 50 µl of the dilute calibration dye into each well of the 96 well Thin Wall
PCR plate (catalog number 223-9441).
4. Press a piece of the iCycler iQ Optical Quality sealing tape (catalog number 223-9444)
onto the 96 well plate.
Note: You may reuse your calibration plate indefinitely, but you must put a new piece of
sealing tape on the plate each time and briefly centrifuge it so that the liquid is at the bottom
of the wells without bubbles or condensation on the sides of the tube.

7.2 Cycling the Calibration Plate

1. Insert the prepared calibration plate into the iCycler.
2. From the front panel of the iCycler, program the following conditions (see the iCycler
Instruction Manual, Section 2.3.2).
Cycle Repeats
1 3
2 Hold
3. Initiate the protocol and choose block or algorithm temperature determination.

7.3 Image Optimization

You must first capture an image of the calibration plate using the iQconfig utility. It is
critically important that none of the wells in the image contain saturated pixels. If there
are any saturated pixels at a 640 ms exposure, you may reduce the exposure time to 320 ms,
but do not decrease the exposure time below 320 ms. If saturated pixels still remain at 320 ms
exposures, you must use a more dilute concentration of fluorescein.
1. From the Start menu, select iQconfig or use the Explorer to open the file.
c:\Program Files\Bio-Rad\iCycler\Ini\iQconfig.exe
This brings up the iQ Configuration window and two dialog boxes: the Image dialog box
and the Gain dialog box (see Figure 6.1.)
2. From the menu bar at the top of the screen, select Filters, and then select Positioning. This
brings up the Filter Positioning dialog box (see Figure 6.1).
3. In the Filter Positioning dialog box is a Cam Filter box and a Lamp Filter box. The Cam
Filter box is for positioning the emission (camera) filter; the Lamp Filter box is for
positioning the excitation (lamp) filter.
Calibration Dye
or
500 µl, 400 nM (Dil 2)
or
800 µl, 400 nM (Dil 2)
or
1.0 ml, 400 nM (Dil 2)
Step Temp Dwell Time
1 95
2 60
1 60
18
Water Final Concentration
7.7 ml 15 nM
7.5 ml 25 nM
7.2 ml 40 nM
7.0 ml 50 nM
00:30
00:30
∞