Biology Reference
In-Depth Information
Fig. 3. Lateral view of the injection chamber. On the left: holding capillary used to
block a blastocyst (dark grey). Edges of the glass walls have been rounded by flaming.
The opening can vary from 50 to 100
m. On the right: injection capillary holding
some ES cells. The opening should be not smaller than the diameter of a cell (about
20
µ
m). A view of the tip from the above (as seen on the microscope) is shown below.
Note the shape of the tip. Both holding and injecting capillaries should be bent (about
120 degrees) so that they remain parallel to the bottom of the injection chamber.
µ
ous task. It is therefore advantageous to prepare sets of these tools in advance
and store them until needed.
3.4.2.1. P
REPARATION OF
H
OLDING
C
APILLARIES
1.
Heat the middle of a tube on a flame until the glass starts to melt. Quickly move
out of the flame and pull the extremities of the tube by hand.
2.
Select capillaries with a 2-3-cm long, 0.1-mm-wide tip.
3.
With the help of a diamond tip, cut sharp and flush the end of the tip.
4.
Adjusting the tip near the heating filament of the microforge, slowly melt the
glass until the opening narrows to 1/4 of the original diameter.
5.
Placing the capillary orthogonal to the heating filament bend the last 3-4 mm of
the tip to form a 30
°
angle.
3.4.2.2. P
REPARATION OF
M
ICROINJECTION
C
APILLARIES
1.
Using a puller, pull tubes so that they form a 1-cm long 1-
µ
m wide tip.
2.
Make a small ball of melted glass on the tip of the heating filament of the
microforge.
3.
Place the tip of the pulled capillary on the filament. Heat to a moderate tempera-
ture (when the ball gets a reddish glow) until the needle slightly fuses with the
glass ball. Immediately switch the heating off. The capillary breaks leaving a
sharp and flush opening (
see
Note 10
).
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