May 10, 1990
Terry C. Lairmore
Introduction
The Perkin-Elmer/ Cetus DNA Thermal Cycler automates the polymerase chain reaction (PCR) technique for amplifying specific DNA sequences. A microprocessor allows you to create programs consisting of time and temperature reaction parameters for individual PCR reactions. These programs can be stored in files which may be modified further for different reactions. In addition, files may be linked together, allowing for more complex programs or "soaking" at 4 degrees C after completion of cycling when reactions are performed overnight.
Files belong to one of two categories, incubation files or cycle files. Files 1 thru 4 are default files which have been programmed with typical parameters. These files can carry out the basic functions of the PCR machine, and they serve as template files which can be modified to program a new PCR reaction or to create user specific files for storage.
File Categories for Default Files
Cycle files: Incubations files:
Soak (#1) - maintains one temperature until file Thermo-Cycle (#3) - a PCR amplification
termination, may be linked to end of cycle file. Step-Cycle(#4) - also a PCR amplification
Time Delay (#2) - incubates samples at a single program.
temperature prior to running a cycle file.
These simplified instructions for using the Perkin-Elmer/Cetus PCR machine will include how to access a file, edit the file, run and/ or save the edited file. For a more detailed description of the various default files and programs, refer to the Perkin-Elmer/Cetus operating instructions manual. It is strongly recommended that you read these instructions and precautions in their entirety before
using the thermal cycler.
Operating the DNA Thermal Cycler
1. Turn the power switch to ON on the front of the machine. The cycler will then display the opening prompt:
"THERMO-CYCLE FILE"
STEP to edit file #3
2. Access the default Thermo-Cycle file #3 from the opening prompt by pressing the STEP key. (Other files are accessed by pressing FILE, press the number of the file you want, then press ENTER)
3. Edit the default Thermo-Cycle file #3 to conform to your desired reaction parameters as follows. When you press the STEP key, the display will take you through the parameters you must answer to modify the file.
4. The message or information on the display is what the instrument is prompting for acceptance or change, and the parameter being prompted is underscored by the cursor. Modification of the default Thermo-Cycle file #3 is a simple way to program a reaction and will be used to explain the machine's operation here. When editing file #3, it is easiest to think of editing segments in pairs. The odd segments (1,3,5) create thermal transitions or ramping of the temperature block to the desired temperature, and the even segments (2, 4, 6) create the thermal plateaus for each of the the three basic steps of the PCR reaction (denaturing, annealing, polymerization). Thus, segments 1 and 2 are a pair, represent ramping of the temperature block to the denaturing temperature (seg. 1) and holding the denaturing temperature for the desired time interval (seg. 2), etc. Entering time zero in segment 7 ends the file. As you press STEP thru the default file Thermo-Cycle file #3, you will edit the parameters of each segment to the ones you desire as detailed in the example below.
Example: a PCR reaction with the following reaction conditions:
"Seg 1 Target Temp"
(0-100) 94C with cursor prompt
This is a thermal transition segment. You can type the temperature you wish to reach for the denaturing step by modifying the value indicated by the cursor. In this case, simply press ENTER to accept the default temperature of 94 degrees C. The time parameter now appears on the display:
"Seg 1 Segment Time"
(0-59) O min 1sec
Because you want the transition to occur as quickly as possible, accept the default values of 0 min 1 seconds by pressing ENTER twice.
To edit the second segment:
The second segment is the thermal plateau for the denaturing segment of the reaction. After pressing ENTER as above, the following appears on the display:
"Seg 2 Target Temp"
(0-100) 94C
Press ENTER again to accept the default temperature of 94 degrees C for denaturing. The following now appears on the display:
"Seg 2 Segment Time"
(0-999) 1 min 0 sec
Press ENTER to accept the 1 minute value and the cursor moves to prompt the seconds parameter. Press 1, 0, and ENTER to enter a value of 10 seconds. The value 10 sec appears on the display, and when you press ENTER, the display moves to segment 3. You are now ready to edit segment 3.
Edit segments 3 to 6 with the following parameters:
When you have finished editing 6 segments, you have created three thermal transition/plateau pairs.
Segment 7 is preset to end the file. Accept the zeros in segment 7 and you have terminated the instructions in your file.
Choose the default NO when prompted for Auto Segment Extension by pressing ENTER. The follwing appears on the screen:
"Cycle Count"
(1-99) 25
Press 3, 5, and ENTER to indicate 35 cycles. Now the following appears on the display:
"Link to stored file"
(0-99, 0=SHUT-OFF)
You may now link the file to an already existing file, such as a stored soak file at 4 degrees C (if for instance the PCR machine will run overnight and you wish the temperature block to go to 4 degrees C after completion of cycling) or you may instruct the machine to shut off. Press the number of the file and ENTER to indicate a file to be linked to the end of your cycle file, or press 0 and ENTER to Shut-Off.
The following now appears on the display:
"End Of File"
RUN-STORE-PRINT
If you wish to store this file for future use, press NO to move the cursor to the "Store" choice and press ENTER. You will then be prompted to enter a user number and a file number under which to store the file for later recall.
To run the newly modified file, press ENTER and then START.
The parameters in the default file will return to their original "default" values after the reaction is run, ready to be modified again for subsequent reactions. Methods for linking files to create more complex reactions, overwriting files, using the printer and additional information may be found in the accompanying Perkin-Elmer/Cetus instruction manual.
References:
Instructions: The DNA Thermal Cycler. (Perkin-Elmer/Cetus) December 20, 1988 Revision
(0993-8412R)