PROTOCOL 018: PULLING NEEDLES FOR ZEBRAFISH MICROINJECTION
Borosilicate glass capillaries with filament
O.D. 1.2mm, I.D. 0.94mm
Note: It is possible to use 1.0mm, 0.78mm capillaries by Sutter (Cat BF100-78-10) but the I.D. may make it difficult to load needles.
P87 micropipette puller
Note: Whenever a new filament is installed, a RAMP test must be performed to determine the correct temperature setting for the puller. See Appendix for how to perform a RAMP test.
Place capillary in trough of left capillary holder (puller bars) and slide along trough until it projects just beyond the holder. Tighten set screw just enough to keep capillary in place (See Figure 1). Do not allow capillary to hit the box filament at any time. Failure to prevent this will damage the filament and make your and everyone else’s needles difficult to use or useless.
Figure 1. Placement of capillary
Depress the retainer clip and slide the puller bar forward until it stops. Use the finger grip on the holder to execute this maneuver (See Figure 2).
While holding the left puller bar with your left index finger on the finger grip, depress the retainer clip on the right puller bar and slide it forward until it stops. With your right thumb and index finger, grab both finger grips and hold the puller bars in place (See Figure 3).
Figure 3. Sliding puller bars together. (G): Groove.
While holding the two puller bars with your right hand, slide the capillary through the box filament and onto the groove on the right puller bar. Position the capillary so that it is centered in the box filament and secure it in place by tightening the set screw (see Figure 4). Make sure both left and right set screws are securely clamping the capillary in place without crushing the capillary. Release the puller bar finger grips they should remain in place.
Figure 4. Anchoring capillary to both puller bars.
Select the appropriate program for pulling zebrafish injection needles. Typical parameters are:
· TEMP: 600-610 (Typically this is set to the value obtained from the RAMP test)
· PULL: 55
· VELOCITY: 70
· TIME: 150
· PRESSURE: 300
To select a program without changing the parameters of the existing program, press the RESET button. This brings up a menu with options <0-9>. The zebrafish needle program is usually <0>.
Depress the <Pull> switch. Take care not to place your hands in the vicinity of the puller bars as they will fly apart rapidly once the heating element melts the capillary.
Once needles are pulled, carefully remove them from the puller bars making sure never to touch the box filament.
When to run a ramp test:
1. Using the puller for the first time (does not apply for current instrument)
2. Whenever a new filament is installed
3. Whenever you switch to a different glass
4. Before writing or editing a program
How to run a ramp test:
1. Enter any program number <0-9>
2. Press clear <CLR> to enter control functions
3. Press <0> to not clear all parameter values
4. Press <1> to run a RAMP TEST
5. Install glass capillary and press <PULL>
When a ramp test is executed, the following events take place:
1. The puller increments the HEAT
2. As the HEAT output begins to soften the glass, the puller bars will move apart
3. The HEAT is the turned off when the factory-set Ramp Test velocity is reached
4. The Ramp Test value will be shown on the display