"Transcriptomics & Functional Genomics"
"Update on using the CFX96"
Bio-Rads CFX96 Real-time qPCR instrument
The EUC funded the purchase of this real-time PCR instrument last year. This instrument, the Bio-Rad CFX96 is housed in the Genomics laboratory in Medical Biomics level 2 rm 58. It has advanced capability covering all real-time chemistries including as TaqMan and Sybr Green, it is also capable of HRM and FRET.
One-off training is being provided on an annual basis in October each year, all users wishing to have some general training in real-time qPCR and use of the instrument including BSc project students are encouraged to contact Dr K Laing. Otherwise training will be provided on a personalised adhoc basis as appropriate to the users needs.
Bio-Rads CFX96 Real-time qPCR instrument
Switching on the instrument and log on to the laptop computer
- The CFX is switched on by depressing a single electrical switch to the rear lower right hand side of the instrument and the laptop computer controling the instrument should be booted. The order of these operations are not important, however the instrument must be switched on prior to starting the CFX Manager software.
- Users should log on to the laptop computer using the “Users” profile, no pass word is required for this.
- Initialising the CFX is performed by starting the CFX Manager software. A shortcut to the software operating the instrument is found on the desk top or the program menu. This is identified by the name “Bio-Rad CFX Manager”. Double clicking on this icon will start the software and initialise the instrument.
- On Start up, the user is prompted with a menu giving the options for “Create a new Experiment”, “Repeating an Experiment”, “Open a Data File”, “Open a Gene Study” or “Open User Preferences”. The “Create a new Experiment” option should be selected for all first time use, in which case click “OK”.
Protocol set up
- A new tabbed “Experiment Setup” window opens within the main panel. The protocol tab is displayed. This window shows the currently selected protocol and at start up this will be the default.
- Click on “Create New” button, a new window will open allowing editing of the protocol.
- This should be performed as follows:
- Temperature and duration of individual steps in the protocol can be altered by firstly clicking on the step to be altered which will then be highlighted with a pale blue background. The temperature or time duration settings for that step may be altered by clicking on the numerical values for the highlighted step in the listing of the steps in the lower planel and entering new values. For these to take effect, it is necessary to select another step. Hours, minutes and seconds are separated by a colon “:”.
- To insert a step at an appropriate place within the amplification protocol select one of the two options (“After” or “Before”) in the drop down box located below the menu bar next to the text “insert step”. Select the step either before or after depending on the option used above and click the “Insert Step” button located in the lower panel. This step can be edited as before "i".
- To change, add or remove a plate read select the step that is to be modified and click either “Add Plate Read” or Remove Plate Read” button and the camera icon will be either added or removed from the selected step
- To insert or change the number of repetitions of one or more steps. Insert a GOTO step at the end of the cycle using the insert “After” option see "ii" above and clicking on the “Insert GOTO” button.
- To edit the GOTO cycle perform the following: The steps that the cycle will include can be altered by selecting the number above the red arrow and changing this to indicate the step at which the cycling will commence. The number below the red arrow indicates the number of repetitions after completion of the 1st cycle and can be edited as for any element of the protocol by clicking on this value and altering it then clicking elsewhere within the window as in "i" above.
- To insert a melt curve (which should always be included in a Sybr Green or similar assay) select the last step in the protocol and click on the “Insert Melt Curve” button. The start, end temperatures and temperature increments and speed of increment can all be altered in the same way as in "i" above.
- Any step may be deleted by first highlighting the appropriate step and clicking on the “Delete Step” button.
- A temperature gradient from Row H to A can be introduced as for any insert step but by clicking on the “Insert Gradient” button. The upper and lower limit of the temperature gradient and duration of the step may be set as with any step ("i" above).
- The individual Reaction volume in each well or tube should be entered in the text box below the menu bar labelled “Sample Volume” and should be an integer between 10 and 25.
- Click “OK” the user will be prompted to save the protocol to file, Click yes and navigate to a users own folder desk top. If one does not exit this should be created using the users name and the protocol file saved to that location with a file name in the format “[ddmmyyyy]_[number of steps in the PCR stage of the protocol]Step[with/without]MeltCurve[optional gene or experimental identifier].prcl” .Click “Save” and the window will close returning the user to the tabbed Experiment Setup window.
Plate Set up
- Now click on the “Plate” Tab. This will display the default plate settings and well locations within the thermocycler block with numerical column values (1-12) across the top and alphabetical row values (A-H) along the side.
- To edit these settings click on the “Create New” button.
- Click on the “Select Fluorophores” button this brings up a table of compatible fluorphores and their respective channels. The appropriate fluorophores used should be selected
- The default scan mode for the CFX is “All Channels” this should not be changed for any real-time PCR application.
- Select all well locations used, then in the options on the right hand side select the respective “Sample Type” (Unknown, Standard, NTC Positive Control, Negative Control or NRT), “Target Name” (eg Gene/Transcript or other target name), “Smaple Name”, “Replicate #” (replicate identifier) or concentration in the case of a standard are entered using free text or from drop down lists should be entered. This may also be performed and altered retrospectively.Making sure the respective “load” check box are checked.
- Experimental settings identifiying normalisers such as normalising genes and / or samples are identified by entering this information into a table following clicking on the “Experiment Settings” button and checking the appropriate check boxes.
- The plate type (clear or white plastic) should be selected according to that used and is located under the menu option “Settings”->”Plate Type”-> “BR Clear “or “BR White”
- “Well Groups” describe locations within the thermocycler block that contain descrete sample sets that should be statistically treated and analysed by the software independently are analogous to separate experiments run in parallel. Well groups can be added by clicking on the “Well Groups” icon under the menu bar. This will open a well grouyp editor and by clicking “Add” and highlighting wells followed by “OK” wells can be added to a well group. Individual wells can belong to multiple well groups
- Click “OK” the user will be prompted to save the protocol to file, Click yes and navigate to a users own folder desk top. The plate file should be saved to that location with a file name in the format “[ddmmyyyy]_[number of steps in the PCR stage of the protocol]Step[with/without]MeltCurve[optional gene or experimental identifier].pltd” .Click “Save” and the window will close returning the user to the tabbed Experiment Setup window.
- Now click on the “Start Run” Tab. This will display the instrument status (Idle), selected Protocol and Plate files.
- The instrument lid should now be opened by clicking on the “open Lid” button“ making should it is safe to do so.
- The Sealed Plate, tube strips or tubes should be loaded into the thermocycler block in the locations specified in the plate layout entered under the “Plate” tab and again making sure it is safe to do do the “Close Lid” button should be clicked.
- When the Lid is closed the “Start Run” button can be clicked to commence the instrument run. The user will be prompted to Open and name the “Run” file. Navigate to a users own folder on the desk top. The plate file should be written to that location with a file name in the format “[ddmmyyyy]_[number of steps in the PCR stage of the protocol]Step[with/without]MeltCurve[optional gene or experimental identifier].pcrd” .Click “Save” and the window will close returning the user to a tabbed Run window. The software will display the remain run time and instrument status as well as graphical output of the Rn values against cycle in real time on one of three tabs in a new window.
- On Run completion the results will be displayed in a tabbed results window. The instrument lid should be opened using the “Open Lid” button displayed and the plate, tube strips or tubes should be safely discared into the appropriate discard container. Again making sure it is safe to do do the “Close Lid” button should be clicked.
- The CFX96 should switched off by depressing a single electrical switch to the rear lower right hand side of the instrument and the Results Window closed by going to “File” ->”Close” and the “CFX Manager” software closed using “File”->”Exit”.
- The run file (xxx.pcrd) should by copied by dropping and dropping the file onto the users flash drive or networked drive.
- The Laptop should shut down using the Start”->”Turn Off Computer” option