25
3.5.3.2
PULL Strength
A constant gravitational pull on the puller bars that can be felt when loading the glass. This
pull is usually adequate to form relatively small tipped pipettes (0.5 mm). Eliminating the
hard pull from the program (PULL=0) is recommended for most patch pipettes. If smaller
tips are required, a moderate PULL (25-50) may be used in the last line of a multi-line
program (see below).
3.5.3.3
VELOCITY (Trip Point)
VELOCITY determines the point at which the heat is turned off. If the value is too high, the
glass will separate after the third heat cycle. As the VELOCITY is decreased, the amount of
glass drawn-out in a given cycle will also decrease, and more cycles will be required to form a
tip. The greater the number of cycles, the larger the pipette’s tip will be and the shorter it’s
taper. However, too many cycles can lead to variability. Generally, it is advisable to keep the
number of heating cycles to 5 or less.
3.5.3.4
Cooling
TIME Mode: When using thin-walled glass to make patch pipettes (slice patch), it is advisable
to keep the TIME between 200 and 250 to maximize the cooling of the glass.
DELAY Mode: The DELAY mode (DELAY = 1) is recommended when pulling a patch pipette
and using thick-walled glass, a box filament, and/or needing short-tapered pipettes. The
DELAY mode provides a set 300 milliseconds of cooling.
3.5.3.5
PRESSURE
The recommended pressure setting when using thick walled glass is 500 or greater. For thin
walled glass, the recommended range is 200 to 500.
3.5.4
Step-by-Step Patch Pipette Programming
Run a Ramp Test with the glass you intend to use for your particular application. Refer to
the manual if you need to review the Ramp Test procedure. When you know the Ramp value
(R), use it in the following program.
1.
Program one line of code as follows:
Filament Type
HEAT
PULL
VELOCITY TIME/DELAY
Box Ramp
0
*30
DELAY=1
Trough
Ramp + 15
0
*50
TIME=150
* The VELOCITY value will need to be manipulated.
PRESSURE should be set to 500 for thick walled glass and 300 for thin walled glass.
2.
Insert your glass and execute the above program. The program should “loop” a multiple
number of times (i.e. the same line will be repeatedly executed). The display will report
the number of loops at the end of the pull sequence. This “looping” is the key to forming
patch pipettes. For thin walled glass, 2 to 3 loops are typically all that is required. For
thick walled glass, 4 to 5 loops are typically required.
3.
Increase the VELOCITY in one unit increments for thick-walled glass and three unit
increments for thin-walled glass. Pull a pipette after each adjustment. Note the change in
P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.43 - DOM (20161118)
Summary of Contents for P-97
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