SOP FOR BIORAD PCR

POWER UP
 Turn on the UPS
 Turn on the PCR and the computer
 Confirm the PCR initializes without errors and displays the home screen
 Launch the Bio-Rad CFX Software
 Confirm the software detects PCR and the PCR screen shows remote
connection

RUN SET-UP
 Select your instrument using the drop down arrow button
 Click ‘User-Defined’ run type to opens the ‘Run Set-up’ window
PROTOCOL SET-UP
 Click on the protocol tab
 For new protocol click ‘create new’. To edit the protocol displayed click ‘Edit
Selected’. The protocol editor window opens
 To use an existing protocol, click ‘Select Existing’, navigate to your protocol
folder and double click the protocol file
 To edit the temperature and time, click on the values displayed and type in
the new values
 To insert step, select a focal step, choose either ‘before’ or ‘after’ using the
drop down arrow, click the ‘Insert step’ command
 To delete a step, select the step and click the ‘Delete step’ command
 To add more details to a step, select the step, click ‘Step options’ command
and update the parameters
 To add plate read to a step, select the step and click ‘Add Plate Read to Step’
command.

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 SOP FOR BIORAD PCR
PLATE SET-UP
A new plate file requires;
(1)Plate size, (2)Plate type, (3)Scan mode, (4)Fluorophore or dye (5)Sample type.
 Click on the plate tab
 For new plate click ‘create new’. To edit the plate displayed click ‘Edit
Selected’.
 To use an existing plate, click ‘Select Existing’, navigate to your plate folder
and double click the plate file
 Confirm plate size. [Settings > Plate Size > 96Wells]
 Confirm plate type. [Settings > Plate type > ?]
 Choose your scan mode using the camera icon on the properties bar; Choose
‘SYBR/FAM Only’ for single channel set-up, or, ‘All channels’ for multiplex set-
up
 Highlight wells you are not using and click ‘Clear Wells’ command to clear the
wells
 Highlight wells of interest and click ‘Select Flourophores’ and select the dyes
or probes for those wells
 Highlight wells of interest and choose sample type using the drop down
arrow
 Highlight wells of interest and type (or select) the target name in the box of
the dye or probe to be used for that target
 Click the 'Load' checkbox to write the target names to the wells
 Highlight wells of interest and type (or Select) a unique sample name for the
well
 Click the 'Load' checkbox to write the sample names to the wells
START RUN
 Click on the ‘Start Run’ tab
 Click ‘Open Lid’ to open the lid and load your samples

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 SOP FOR BIORAD PCR
 Click ‘Close Lid’
 Click ‘Start Run’ to start your run

DATA ANALYSIS
CFX software processes real-time PCR data automatically at the end of each run and
opens the Data Analysis window to display these data (the .pcrd file).
The Data Analysis window displays multiple tabs. Tabs appear only if the data
collected in the run are available for that type of analysis.
 Choose analysis mode. [Settings > Analysis Mode > ?]
 Choose Cq determination mode. [Settings > Cq determination mode > ?]
 Choose baseline setting. [Settings > baseline setting > ?]
 Define the number of cycles to analyse. [Settings > Cycles to Analyse]
THRESHOLD ADJUSTMENT
 Switch to Log scale
 Select only one target
 Drag the threshold line to meet the baseline, OR, goto [Settings > baseline
threshold]
 Enter a value for the ‘single threshold’. Note that your Cq determination mode
must be ‘single threshold’ for you to be able to edit single threshold value

REPORTS
To Print any window of interest, Right-Click on that window and select ‘Print’
For Complete Report of Run, navigate to Tools > Reports. [The reports window
opens]
Select parameters you want to display and click update report.
Save report.

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