Biometra TGRADIENT 48 User manual

TGRADIENT Manual ___________________________________________________________ 1
Manual
Software Version 4.20
May 2003
Order-No.
050-800 TGradient 48
050-801 TGradient 96
Biometra
biomedizinische Analytik GmbH
Rudolf-Wissell-Str. 30, D-37079 Goettingen
Tel.: [phone]; Fax: [phone]
email: [email protected]
http://www.biometra.com

TGRADIENT Manual ___________________________________________________________ 2
1 Introduction
The TGRADIENT is the new high end thermocycler in the Biometra product line. It was our goal
to develop an instrument that provides fast optimization of reaction parameters as well as a high
performance for daily routine analysis. Both could be realized by a completely new block
architecture and the latest Peltier technology.
To increase the specificity of a reaction, the annealing temperature is one of the crucial
parameters. With the new TGRADIENT different annealing temperatures can be tested within a
single run. A gradient with a maximum range of 40°C can be set at any program step. We have
taken strong efforts to create a gradient as linear as possible. At the same time it was very
important to ensure homogenous temperature distribution within each row. The temperature data
for each row of the block can be retrieved through the help menu.
The new TGRADIENT is an outstanding instrument. In addition to the advanced temperature
gradient it provides high speed and reliable accuracy for routine applications. The silver block
features very high heat conductivity and ensures fast ramping speeds and a fast temperature
equilibration.
The new TGRADIENT will speed up your laboratory work because it saves time not only in
optimizing new reactions but also in your routine work.

TGRADIENT Manual ___________________________________________________________ 3
2 Before you start
2.1 Safety warnings
Before connecting the unit to the power source please ensure that the
voltage selector at the back side of the instrument is set to the required
voltage.
Danger of electric shock! Unplug the power cable before you open the
TGRADIENT Thermocycler.
It is not necessary to utilize oil into the opening of the block in order to
improve the heat transfer between the block and the sample tubes. If you
still decide to use oil, do not use silicon oil. Mineral oil may be used.
2.2 Warning symbols
The thermoblock and the heated lid will reach high temperatures
during operation. Both thermoblock and heated lid can burn you.
Rapid heating of the thermoblock can cause liquids to boil
explosively. Always wear safety goggles during operation. Close the
lid before starting a program.
Ensure that both the rear and bottom ventilation slits of the rear and
bottom of the instrument are unobstructed. Insufficient ventilation
can cause overheating of the instrument.

TGRADIENT Manual ___________________________________________________________ 4
3 First steps with the TGRADIENT
3.1 The TGRADIENT Thermocycler front View
Wheel for the height
adjustment
of the heated lid.
Keypad
Graphical display
Power switch
Knob for release
of the lid

TGRADIENT Manual ___________________________________________________________ 5
3.2 The TGRADIENT rear view
Parallel
printer port
RS-232
port
Power cord
plug
Fuse holder
for 2 fuses,
for specifications of fuses
see type label on the
instrument´s backside
Type label including:
serial number
specifications of fuses

TGRADIENT Manual ___________________________________________________________ 6
3.3 The TGradient control panel
798
123
456
.0-
ABCD
Pow er
Cursor keys:
For free navigation in the
programming spreadsheet.
Use to scroll through lists.
Function keys:
the corresponding functions are
specified in the screen.
Power switch:
backlit = power on
Easy to clean
keyboard
Graphical display

TGRADIENT Manual ___________________________________________________________ 7
3.4 Initial self test
After switching on the TGRADIENT the serial number of the instrument and the software version
is displayed.
TGradient
Serial No 1234567
Vers. 4.00 op
The TGRADIENT checks all programs in all directories (so called RAM check).
3.5 The TGradient display
The TGRADIENT display provides information about the instrument and about the status of the
active program.
prog
step
temp[°C] 25.2°C
time
lid 25.4°C
A ? B start/stop C programs D +
The individual function of the special keys A , B , C and D are specified in the display and
depend on the context.
3.6 Handling of the adjustable heated Lid
To achieve optimum pressure on the tubes the TGRADIENT is equipped with an height adjustable
heated lid.
Close the lid:
After the samples have been placed in the block close the lid. Turn the wheel clockwise until you
hear a clicking noise. In this mode the pressure will not increase further, even when you keep on
turning the wheel.
Note: The pressure of the lid has been optimize for a fully loaded block. If only very few tubes
are loaded to the block you should place dummy tubes in the four corner positions to avoid
damage of tubes by excessive pressure.
Open the heated lid:
First: Release pressure by turning the wheel counter clockwise. As soon as there is no more
resistance the pressure has been released. Then: now you can open the lid with the knob.
Important: The lid should not be opened under pressure because this leads to damage of the
locking mechanism.

TGRADIENT Manual ___________________________________________________________ 8
3.7 Releasing wheel in case of blocked lid
Note: When the lid is in the extreme up or down position, it may happen that the wheel is
uncoupled. In this situation the clutch mechanism is active in both directions (clicking noise in
either direction). To unlock wheel, press metal pin with a ballpen and turn wheel carefully. This
pin overcomes the automatic clutch mechanism. Thus, care must be taken not to apply excessive
pressure.
Lid arrested in upper
position:
carefully turn wheel with
pressed pin clockwise, until
you feel normal resistance (no
more clicking noise, clutch is
released). Release pin and
turn lid down, until the clutch
mechanism is activated
(clicking noise, optimum
pressure applied).
Lid arrested in down
position:
carefully turn wheel with
pressed pin counter
clockwise, until you feel
normal resistance (no more
clicking noise, clutch is
released). Release pin and
turn wheel counter clockwise
until pressure is completely
released. Open lid.
IMPORTANT: WHEN THE CLUTCH MECHANISM IS ACTIVE (OPTIMUM
PRESSURE IS APPLIED), NEVER USE PIN TO FURTHER INCREASE PRESSURE.
THIS WILL LEAD TO DAMAGE OF TUBES AND INSTRUMENT!

TGRADIENT Manual ___________________________________________________________ 9
4 Creating a Program
4.1 Select a directory
In the TGRADIENT programs can be stored both in the main directory and in individual
subdirectories. For easy identification of the selected storage place, the subdirectories can be
named (see section 4.3).
Start with the main screen.
Prog
step
temp[°C] 25.2°C
time
lid 25.4°C
A ? B start/stop C programs D +
Press [C programs] to enter the edit mode.
½-3. subdirect.:
½-4. subdirect.:
Main direct.--- ½-5. Subdirect.:
½-6. subdirect.:
½-7. subdirect.:
A ? B C quit D enter
You are now in the main directory. The structure of the subdirectories is displayed.
4.2 Select a program store
Programs can be stored in the main directory or in subdirectories.
To create a program in the main directory press [D enter].
To access a subdirectory move right with the cursor key ðand select a subdirectory with the
cursor keys ñò. The selected directory will be highlighted.
½-3. subdirect.:
½-4. subdirect.:
Main direct.--- ½-5. Subdirect.:
½-6. subdirect.:
½-7. subdirect.:
A ? B C quit D enter

TGRADIENT Manual __________________________________________________________ 10
4.3 Enter name for subdirectory
The process for naming a subdirectory and a program is the same. For information about naming
programs see section 4.4.
Press [D enter] to enter the subdirectory
directory: 5
program no. Name:
edit
A list B C quit D enter
Press [A list] to view a list of all existing files and empty memory in this directory.
Note: If neither program name nor the word “empty” is displayed, this place may be occupied by
a program without name. For the programming of new protocols choose an empty memory.
Existing programs that are not needed any more can be deleted to provide new memory.
0 empty
1 empty
2 empty
3 empty
4 empty
A list B forward C quit D enter
Scroll through the list with cursor keys ñò and confirm memory with [D enter].
4.4 Enter a program name
Each program is specified by a program number and a subdirectory number. To make retrieval of
a program easier, you can enter a name for each program existing of letters, numbers and
symbols.
Directory: 3
program no. 0 Name:
Ù
ABCDEFGHIJKLMNOPQRST
UVWXYZ-()#°C/,áñ&+.%!
A ABC B files C name OK D enter
To enter a name for your program press [A ABC].
Directory: 3
program no. 0 Name:
Ù
ABCDEFGHIJKLMNOPQRST
UVWXYZ-()#°C/,áñ&+.%!
A name B blank C quit D enter
Choose a letter from the alphabet with cursor keys ïðñò

TGRADIENT Manual __________________________________________________________ 11
Confirm each letter with [D enter].
Directory: 3
program no. 0 Name: T
Ù
ABCDEFGHIJKLMNOPQRST
UVWXYZ-()#°C/,áñ&+.%!
A name B blank C name OK D enter
The selected letter will appear in the display and the cursor will move one step to the right.
Select the next letter and confirm with [D enter].
Note: What to do if a wrong letter has been accepted?
To alter the position of the cursor within the name press [A name]. Now you can move the cursor
with cursor keys ïð through the name. This is helpful if you have confirmed an wrong letter or
if you want to change the name of an existing program.
Once the name is complete, confirm name with [C name OK].
Directory: 3
program no. 0 Name: TEST 1
lid temp: °C preheating: on
edit
A ? B files C pgm OK D enter
4.5 Enter the lid temperature
You can now enter a temperature for the heated lid. Note: In general, the lid temperature should
be about 10°C above the highest temperature in the protocol. Example: If your denaturation step
is at 95°C, enter 105°C for the lid.
Directory: 3
program no. 0 Name: TEST 1
lid temp: 105 °C preheating: on
edit
A ? B files C pgm OK D enter
4.6 Select / deselect pre-heating of the lid
You can choose whether the lid is pre-heated before the program starts. This is to avoid
condensation during the initial heating phase. Note: During the preheating of the lid, the block is
held constant at 25°C.

TGRADIENT Manual __________________________________________________________ 12
Directory: 3
program no. 0 Name: TEST 1
lid temp: 105 °C preheating: on
edit
A on/off B files C pgm OK D enter
Note: The default setting, which is recommended for most applications, is preheating: on.
After all pre-settings have been made, press [D enter] to open the programming spreadsheet.
4.7 Enter temperature and time settings
The programming spreadsheet:
Temp[°C] time ß# gradient[°C] opt à
1
2:
3:
4:
A ? B insert/delete C pgm OK D enter
Note: In this spreadsheet you can enter all parameters for your cycling protocol. Furthermore,
you can set special parameters like touch down, ramping rates or time increments. For further
details on these special parameters, see section 5.1.
Note: You can navigate in the spreadsheet without limitations with the four cursor keys
ïðñò.
Note: Each setting is confirmed with [D enter]. The cursor moves automatically to the next field.
Alternatively, you can confirm a value by moving forward with the cursor keys.
Note: At any time you can call up the help function with [A ?]
Note: In an existing protocol program steps can be deleted and inserted. For further information
on deleting and inserting program steps see section 5.1.
Now enter temperature for the first step in the protocol:
Temp[°C] time ß# gradient[°C] opt à
1 95,0
2:
3:
4:
A ? B insert/delete C pgm OK D enter
Confirm temperature with [D enter] or just move on with the cursor key. If you move to the next
row, the setting will be stored in the temporary memory.

TGRADIENT Manual __________________________________________________________ 13
Temp[°C] time ß# gradient[°C] opt à
1 95.0
2:
3:
4:
A ? B insert/delete C pgm OK D enter
Enter a time for step one.
Note: There is a convention on how time settings are entered in all BIOMETRA cyclers:
hours lminutes lseconds
If you enter a number without “point” this value will be interpreted as seconds (“300” => 5
minutes). To program minutes enter a “l” after the number of minutes. To enter hours enter ll
after the number. You can also enter any combination of hours, minutes and seconds. Example:
for 1 hour, 30 minutes, 20 seconds enter 1l30 l20.
The time values will be displayed in the following format: 0h 00 m 00s
Accept time setting with [D enter] or move to the next field with cursor key.
4.8 Set temperature gradient
Note: Each temperature gradient is defined by two settings: first: the temperature in the middle
of the block, second: the range of the gradient. For example if you want to program a
temperature gradient from 50 to 60°C, enter 55°C as temperature an 10°C for the gradient.
Temp[°C] time ß# gradient[°C] opt à
1: 95.0 0h 5m 0s
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s 10°C
4:
A ? B insert/delete C pgm OK D enter
Note: The maximum temperature gradient is 40°C.
Note: You can program as many steps with temperature gradient as you want.
Note: You can even set an inverse temperature gradient. An inverse gradient has the higher
temperature on the left side of the block and the lower temperature on the right side respectively.
Inverse gradient are set by adding the algebraic sign “-“ to the gradient value.
4.9 View different temperatures of the gradient in the block
To view the temperatures in the 12 different block rows press [A ?]

TGRADIENT Manual __________________________________________________________ 14
Temp[°C] time ß# gradient[°C] opt à
T1 – 6: 50.0 50.2 50.9 52.0 53.2 54.4 °C
T7 –12: 55.6 56.8 58.0 59.1 59.8 60.0 °C
3: 55.0 0h 0m30s 10°C
4:
Temperature difference left side / right side
In the first highlighted lane the temperatures from row 1 to 6 are displayed. In the second
highlighted lane the temperatures from row 7 to 12 are displayed.
Note: While the TGRADIENT is cycling you can view the current gradient temperatures. The
temperatures can be displayed online during each gradient step (see section 7.4). As the program
moves to the next step the last gradient temperatures are stored. Thus you can check the
temperatures of the last gradient in consecutive steps without gradient. For further information
on the current status of the cycler see section 7.
4.10 Set cycle number
Note: In general, loops are defined by selecting the target for the back loop and the number of
backloops.
Temp[°C] time ß# gradient[°C] opt à
1: 95.0 0h 5m 0s
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2
A ? B insert/delete C pgm OK D enter
In the field labeled with ßyou can select the target step for a backloop.
Temp[°C] time ß# gradient[°C] opt à
1: 95.0 0h 5m 0s
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
A ? B insert/delete C pgm OK D enter
Enter the number of backloops. Note: total cycler number = (n backloops) + 1, e.g. enter 29 for a
total number of 30 cycles.
4.11 Cool below ambient temperature
Temp[°C] time ß# gradient[°C] opt à
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
5: 4.0 pause
A ? B insert/delete C pgm OK D enter
To hold the temperature for an indefinite time enter “0”. After pressing [D enter] “pause” is
displayed.

TGRADIENT Manual __________________________________________________________ 15
Note: The minimum temperature you can set is –3°C.
4.12 Save program
Save program with [C pgm OK]. The program data are stored in the permanent memory.
Directory: 3
program no. 0 Name: TEST 1
number of steps: 5
runtime: 1h10m23s
edit
A B C D
The runtime is calculated and displayed.
Note: To view the current remaining runtime at any time of the run press [A info] in the main
menu.

TGRADIENT Manual __________________________________________________________ 16
5 View/edit program data
You can check any existing program by scrolling through the editing mode with cursor keys
ïðñò. Select the desired program as described in section 4.2.
Note: During operation of the cycler the active program can be viewed but not modified. If you
want to change settings of the active program for further experiments you have to save a copy of
this program to another memory. For further information about copying programs see section
5.2.
5.1 Delete / insert program steps
The programming spreadsheet:
Temp[°C] time ß# gradient[°C] opt à
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
5:
A ? B insert/delete C pgm OK D enter
To delete or insert a program step press [Binsert/delete]
insert / delete step:
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
5:
A ? B delete input C quit D enter
Enter the number of the step that you want to insert / delete.
Note: Additional steps will be inserted before the selected step number. The following steps will
be shifted by one position.
insert / delete step: 3
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
5:
A ? B delete input C quit D enter
Note: If you have entered a wrong number, the field can be cleared with [B delete input].
Confirm the step number with [D enter].

TGRADIENT Manual __________________________________________________________ 17
insert / delete step: 3
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2 29
5:
A insert B delete C quit D enter
Press [A insert] to insert one step at the selected position.
Note: Additional steps will be inserted before the selected step. The following steps will be
shifted by one position.
Press [B delete] to delete the selected step.
Note: By deleting a step the following steps will be shifted by one position.
Note: The insertion and deletion of steps may have an influence on existing cycles. Be sure that
all settings are updated to maintain a correct cycle.
5.2 Copy program
Select the program you want to copy as described in section 4.1 and 4.2
Directory: 3
program no. 0 Name: TEST 1
lid temp: 105°C preheating: on
A ? B files C pgm OK D enter
Press [Bfiles].
Directory: 3
program no. 0 Name: TEST 1
lid temp: °C preheating: on
edit
A copy B delete pgm C quit D enter
To copy this program to another memory press [A copy]
copy to: ½-3. subdirect.:
½-4. subdirect.:
Main direct.--- ½-5. Subdirect.:
½-6. subdirect.:
½-7. subdirect.:
A ? B C quit D enter
Select the desired subdirectory with the cursor keys ðñ ò. The selected subdirectory will
highlight.

TGRADIENT Manual __________________________________________________________ 18
copy to: ½-3. subdirect.:
½-4. subdirect.:
Main direct.--- ½-5. Subdirect.:
½-6. subdirect.:
½-7. subdirect.:
A ? B C quit D enter
Confirm your selection with [D enter].
copy from directory 3 program 0
to directory 6 program
A list B C quit D enter
Choose an empty memory from the list with [A list].
copy from directory 3 program 0
to directory 6 program 6
enter = OK
target program contains 0 steps
A list B C quit D enter
Note: Before you confirm with [D enter] the target memory is checked. If there is no program in
the target, memory the message “ target program contains 0 steps” is displayed. If the memory
already contains a program the number of steps is displayed.
Note: If you copy a program to a memory that already contains a program, the former program
will be deleted!
Save a copy of the program in the new directory with [D enter].
5.3 Delete program
Select program as described in section 4.1 and 4.2
Directory: 3
program no. 0 Name: TEST 1
lid temp: °C preheating: on
edit
A copy B delete pgm C quit D enter
To delete this program press [Bdelete pgm].

TGRADIENT Manual __________________________________________________________ 19
Directory: 3
program no. 0 Name: TEST 1
lid temp: °C preheating: on
delete program 0? enter = yes
A copy B delete pgm C quit D enter
Confirm your selection with [D enter].
Note: Once you have deleted a program it cannot be restored!

TGRADIENT Manual __________________________________________________________ 20
6 Further programming options
Temp[°C] time ß# gradient[°C] opt à
1: 95.0 0h 5m 0s
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s
4: 72.0 0h 0m30s 2
A ? B header C pgm OK D enter
Note: To program special parameters you can scroll the display to the right.
Move the cursor to the right side of the display that is labeled with opt à.
The following screen will appear:
Main funct. dT[°C] dt[s] ä[°/s]
1: ß5.00
2: ß5.00
3: ß5.00
4: ß5.00
A ? B header C pgm OK D enter
Note: To get back to the screen with the basic parameters move the cursor to the left side of the
display labeled with “Main funct.” Alternatively you can move the cursor to the extreme right.
Temp[°C] time ß# gradient[°C] opt à
1: 95.0 0h 5m 0s
2: 95.0 0h 0m30s
3: 55.0 0h 0m30s +
4: 72.0 0h 0m30s 2
A ? B header C pgm OK D enter
Note: In the main screen you will find an information that a special parameter has been
modified. This is indicated by a + in the row labeled with opt à.
6.1 Program time increments
To compensate for loss in enzyme activity each step within a loop can be extended from cycle to
cycle. Enter the desired time increment [seconds] in the row labeled with dt[s]. This value will
be added to the time value from cycle to cycle.
Main funct. dT[°C] dt[s] ä[°/s]
2: ß5.00
3: ß5.00
4: ß10 5.00
5: ß5.00
A ? B header C pgm OK D enter