Perkin-Elmer Thermal cycler (2400/9600)

Turn on thermal cycler – switch on left front.

Samples
1. for 2400 machines, place samples in holder. Open holder, place tubes in holder. Snap top in place. Put caps on tube. There are some tubes + caps that don’t have to be separated, just make sure that the tubes sit low enough that the lid closes properly.
2. for 9600 machines, place plate firmly into machine. Place caps on all samples. Use punch side of black tool to push caps down firmly.
Create Method
1. F2 (Create) – opens a basic method.
2. Using arrows move from cycle list to temp. to time; type in changes and then press Enter.
3. F2 (Store)
4. Change name – F3 (Method)
5. Backspace (F3) to erase the default name. Use arrows to move along alphabet. Enter/Accept/Accept
User
1. Select F5 (User)
2. F2 (new)
3. use arrows to select letters of name
4. accept
5. Enter a pin # if you want (default – none)
6. Accept
Edit method
1. F3 (Edit)
2. Select method to edit (use arrows to highlight method you want to edit).
3. You can view the method before editing if you want; F2 (View)
4. F1 (edit)
5. Make any changes you want – the same way as creating new method.
6. Store/Accept
Run
1. F1 (run)
2. Select (highlight) method you want to run
3. F1 (start)
4. enter volume in tubes/plate
5. Start
Stop Run
1. to end method, press Stop button
2. press Stop button again
3. Select Exit.
4. Turn off thermal cycler
Mastercycler Gradient Thermal Cycler

To Edit an Existing Program and Save as a New Program
1. Turn on (Power in back left hand side).
2. Main menu; use Arrows; go to Files; Press Enter.
3. Arrow to Load; press enter.
4. Arrow to choose program you want to edit; Press Enter. Sometimes when you press Load the list of programs doesn’t come up, it only asks you to save the previous program. Press SEL to change the Yes to No and press Enter. If this doesn’t work the first time, repeat.
5. Arrow down to parameter line that you want to change.
6. Arrow over to part of the parameter line you want to change. Use keypad to make changes, press Enter.
7. DEL: deletes the line that the cursor is on.
8. INS: to insert a line above the cursor.
9. When done, press Exit.
10. Save: Yes. Press Enter to save; it you do not want to save, press SEL to change the highlighted Yes to No and press Enter.
11. Change name – use a name and a code to identify your file.
To Start a Run
1. Turn on (Power in back left hand side).
2. Arrow to Start; press Enter.
3. Scroll to file you want; press Enter.
4. Program is automatically tested and starts. Press OPT to see finish time.
To Stop. Press Start/Stop and then Enter.
To set up a Standard PCR program
1. Select Files; Press Enter.
2. Select standard; press Enter.
3. Change parameters using the arrows to move the cursor and then make changes using the keypad, Press enter before changing another line. See editing and existing program above to add or delete lines.
4. At the end of the program: Go To – select which line to go to for multiple cycles (usually step 2, after the initial longer denaturation step).
5. Set Rep – select the # of cycles to repeat.
6. Add a Final Extension step if you want to. This is not included in the Rep cycles.
7. SEL, select until find Hold. Enter temp. you want your samples to hold until you get them – usually 4oC.
8. Exit.
9. Save: Yes; press Enter.
10. Change name – use your name and a code to identify file.
To set up a Gradient PCR program
1. Select file; press Enter.
2. Select New; press Enter.
3. Move cursor to lid using arrows; move across to lid and type in 105; press Enter. Leave No Wait and Auto.
4. Arrow down; SEL and a line for Temp. and Time appears above end.
5. Initial Denaturation. Enter Temp. and move cursor across to enter Time. Press Enter.
6. Denaturation. SEL. Enter Temp and Time and press Enter.
7. Gradient. SEL. Enter Temp and Time. Press Enter. Move cursor back up to line you just entered. Select OPT and 5 additional options appear. Arrow up and arrow over so that cursor is under Temp. and then arrow down to the 5 options. If you don’t move the cursor under Temp. before going down, the gradient parameters will disappear. Arrow down to G = __ oC. The only one you need to change is G = 0.0o. The other options are: +0.0o is for temperature increment, the temp. for each cycle is increased or decreased by the value entered; +0:00 is the time increment, for each cycle the time is extended or reduced by this value; R= 3o/s is the ramp value, how fast the cycler ramps up or down, it is usually set at 3o/sec.; G = the gradient, each column is set at a different temp., the center of the block is the Temp. entered at the beginning of the parameter line. G tells the thermal cycler how much of a spread across the block the gradient will go with the lowest temp on the left and the highest temp. on the right. For example, G = 10oC, with a Temp. of 50oC means that the gradient will spread 10 oC above 50oC and 10oC below 50oC. with column 7 closest to 50oC.
8. Extension. Add an Extension step the same way you entered the denaturation step.
9. SEL until Go To appears in the parameter line. Enter which line to go to for multiple cycles (usually step 2, after the initial longer denaturation step). Set Rep – select the # of cycles to repeat. Press Enter.
11. Add a Final Extension step if you want to. This is not included in the Rep cycles. Press Enter.
12. SEL, select until find Hold. Enter temp. you want your samples to hold until you get them – usually 4oC. Press Enter.
13. Exit.
14. Save: Yes; press Enter.
15. Change name – use your name and a code to identify file.
To see Temp. Range of Gradient
1. Main menu; select OPTIONS, press Enter.
2. Select Gradient; press Enter
3. Change parameters to match your run. For example: Tube Size = 0.2 ml; T = 50oC; G = 10oC. Press Enter.
4. No printer so you have to write temp. for all 12 rows down. You also can’t access this function during the run. Be sure to put in gradient range – enter 10 (for 10 above and 10 below middle temp).

Hint for gradient run: to find optimal annealing temp. Set up tubes in every other row. You will still get a nice temp. range, but will save template and reagents.

This information is given as a guide to the facilities and instrumentation available in the DNA/Protein Core facility at Georgia State University. If you have any concerns or thoughts about the content of this website please contact: John Houghton (404) 413-5390