Multichannel Systems Roboinject Manual - Manual (Page 84)

Roboinject Manual
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6.5.6 Plating Oocytes
You need a well plate filled with Barth's + gentamicin.
Important: Visually check all well plates before use. Use only unsterile and very flat plates.
Sterilization may lead to warped plates. Warped plates will decrease the injection and
expression efficiency.
1. Aspirate a number of oocytes with a transfer pipette by applying a low negative pressure,
for example, with a rubber ball.
2. Move from well to well with the transfer pipette, carefully dropping one oocyte into each
of the wells of the well plate. Grip only the glass pipette and do not squeeze the rubber ball,
to avoid that too many oocytes will leave the pipette. The oocyte should drop into the fluid
by its weight alone.
3. If occasionally more than one cell drops into a well, this can be corrected after the plating
will have been finished.
4. Due to the higher density of the vegetal pole, the oocytes will usually settle with the animal
pole facing up if the (sticky) follicle cells were completely removed. Immediately check the
position of each oocyte under a stereo microscope, and correct it carefully with the golf-club
shaped glass tool, if the orientation is not correct. A manual correction should be necessary
for less than five percent of the oocytes if the follicle cells were completely removed.
5. Cover the well plate with its lid, and seal the rim with Parafilm to reduce evaporation,
and store it at 18 °C until injection.
6. It is possible to inject the cells right after plating, but the oocytes should not be further
disturbed, for example not be washed or otherwise treated until they have been allowed to
adhere to the well bottoms (about 2-3 hours). The oocytes can also be injected on the next day,
after being stored overnight at 18 °C. If injected on the next day, it is recommended to check
the viability again under the microscope, and replace bad oocytes before the injection.
Please note that oocytes sometimes express better when they are fresh and not stored
overnight before injection.