Vous êtes sur la page 1sur 2

QuickStart Guide to Operation of Bio-Rad iQ5 (qPCR)

Startup 1. Turn on the computer, 2. Turn on switches for both the iCycler (PCR machine, bottom right) and the iQ5 (detection system, top right). 3. Launch the data acquisition software with the Bio-Rad iQ5 icon on the desktop. The opening window is divided into Workshop, Run Time Central, Data Analysis and Calibration modules on the left, with the Workshop module active. On the right you will see quadrants with file navigator, data, protocol and setup windows.

Setup 4. In plate setup, click Create New, then Select/Add Fluorophores to specify reporter dyes for monitoring. You can also open a previously saved data file and edit the setup information contained therein. Note that data files contained linked setup and protocol files. Click OK to exit. 5. Select sample, standard, etc. from the well definition icons along the top of template and click-drag to specify, using adjacent columns in one row for duplicates. 6. Click spreadsheet to enter identifiers for samples or values for standards. 7. Save setup. This takes you to the User1 folder; create a folder and save a .pts setup file. 8. By running each sample, whether gene of interest or reference, at a minimum of three dilutions, each in duplicate, you will be able to calculate amplification efficiencies, allowing use of Pfaffls method (Nuc. Acids Res. 29(9): p. 2002. 2001) for data analysis, which corrects CTs for differing amplification efficiencies.

Protocol 9. To open a previously saved Setup or Protocol file, select folder in file tree and then click either Plate or Protocol to display the appropriate file types. In the Protocol quadrant select Create New or Edit. A bare bones thermal cycler setup comes up. 10. Use the Insert or Delete keys to add or remove Steps to a Cycle, positioning the cursor and using the delete key to edit temperature and/or time values. Specify data collection steps by adding real-time from the drop down menu; a camera icon shows up on this line. The minimum dwell time (for data acquisition) is 10 sec per fluorophore. 11. When Gradient is selected from the Show Options box, a two-column spreadsheet opens in which the row temperatures can be set. 12. Use the infinite hold time to drop the thermal cycler temp at the end of a run. 13. A Melt Curve cycle can be added to the end of the amplification cycles in the protocol, or it can be set up as a separate protocol. Select Melt Curve under the PCR/Melt Data Acquisition column, enter the appropriate number of repeats, the dwell time, etc. 14. Save the .tmo protocol file to your folder.

Initiate Run

15. Once setup and protocol files have been loaded and edited, click Run in the upperright quadrant. A save dialog window opens; navigate to your folder and enter a file name. Select persistent or dynamic for well factor collection. With fluorescein in wells, use dynamic. These factors define the addresses on the CCD chip for each of the 96 wells. Upon initiating the run you a Monitor Run window opens automatically. The amplification plots will be diplayed during the run (the ABI 7700 differs, in that the plots can be displayed only at the end of the run).

Data Analysis 16. At the end of the run data will be displayed in the Data Analysis module. Click PCR Quant to display the amplification plots, standard curve and spreadsheet with well positions, definitions and CTs. Bio-Rad does not use normalization with a reference dye in processing raw signal, an option found in data analysis after acquisition with the ABI 7700. 17. Adjust the cycles used for baseline calculation by right click in the amplification plot window and selecting Baseline Threshold. This opens a dialog box in which the cycles can be selected automatically or manually defined by the user. 18. Adjust the threshold in log view, and note that CTs are automatically recalculated. 19. Columns on the spreadsheet can be selected by clicking the headers, copied and pasted into Notepad or Excel, then saved to your flash drive for later analysis.