WPI MICRO-ePUMP, Инструкция по эксплуатации

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MICRO-ePUMP
World Precision Instruments 19
The following table is useful for converting between different units of volume.
Cubic Measure 1 cm
3
1 mm
3
100 µm
3
10 µm
3
1 µm
3
Volume 1 mL 1 µL 1 nL 1 pL (10
-12
L) 1 fL (10
-15
L)
For ejected volumes less than 1 nL, visual inspection in air proves to be difficult due
to rapid evaporation. The same technique may be used though if the drop is kept
submerged under oil. Droplets may seem to disappear after emergence from the tip.
Sometimes this is due to creepage of the aqueous fluid back along the outside shank of
the micropipette. This creepage may be decreased by silanizing the outside of the pipette.
TIP : Precise assays of ejected volume may also be obtained through various
radioisotopic methods. See the bibliography for further information.
A slight deflection of the micropipette tip may be noticed during the application of the
pressure pulse. This deflection may be eliminated by ensuring that the micropipette is
firmly seated in the holder and that the holder is firmly held by a manipulator.
Calibrating Rate using a Known Volume
We can calculate the volume of liquid contained in a 1 mm length of a glass pipette, if
we know the inner diameter (ID). Then, we can inject that known volume and measure
the time it takes to inject it. From this we can calculate the rate of injection. For
example, if the volume in 1 mm of glass with 0.58 µm ID (WPI # 1B100 ) is 264 nL, and
it takes 30 seconds to inject that volume, then the flow for 1 second is 264 nL/30 s or
8.8 nL/second, and a 1 nL volume then takes 0.113 seconds to inject.
This method is discussed in the following JoVE video: http://www.jove.com/video/2079/
intravenous-microinjections-zebrafish-larvae-to-study-acute-kidney.
In the Jove video, the capillary tip is 10–20 µm. By following these steps, we are able to
calculate the timing needed to deliver a 1 nl injection by counting the time between 1
mm marks as the fluid is injected.
NOTE : The MICRO-ePUMP timing should not be adjusted to go below 10–15 ms as an
absolute minimum, since it takes 6–10 ms for the pressure valves to respond.
1. Fill a small dish with mineral oil and place it under a stereo microscope.
2. Turn on your air and vacuum pumps, and set your compensation pressure and
your injection pressure. The proper settings must be determined experimentally.
Turn on the MICRO-ePUMP .
NOTE : The injection pressure must exceed the compensation pressure in order
to eject the fluid.
3. Use a pipetter to inject a 10 µl sphere of fluid into the mineral oil.
4. Using a fine pointed permanent marker, mark the injecting micropipette at 1 mm
increments.
5. Mount your “graduated” micropipette on the MICRO-ePUMP that is connected to
the Injection Port on the MICRO-ePUMP .
6. Set the mode to Manual. Hold down the foot switch to inject fluid from the
micropipette into the oil-filled dish.