Eppendorf 012: Microinjection of plasmid DNA or double stranded RNA - Manual (Page 3)

Description
Programmable microinjector with external
pressure supply
Propo rtional micromanipulator for microinjection
Dynamic micromanipulator for microinjection,
can be combined with FemtoJet express
Dynamic micromanipulator for microinjection
Special pipette tips for filling Femtotips from
the rear
0.5 – 10 µl
Injection
The parameters (injection pressure (Pi), compensation pressure (Pc), injection time (Ti)) of the FemtoJet express microin-
jector should be adjusted according to the quality of the needle and how the needle is broken. There aren´t any fixed
parameters for injection pressure nor for the duration of injection.
The FemtoJet express offers an "automatic" and a "manual" injection mode. The injection is triggered by hand control
or an optional foot control. "Automatic" means that the duration of injection is preset on the device by defining the injec-
tion time (Ti). In the "Manual" mode the injection time is determined individually, which means that injection proceeds as
long as the foot or hand control is triggered. In our laboratory we use this mode as injection is performed until some
liquid can be seen in the gonads.
Inject into the mitotic region of the worm gonad by gently pushing the microscope stage (the worm is moving accor-
dingly) towards the needle and carefully move the micromanipulator towards the worm. Inject the mixture by pressing the
left mouse button. The success of injection can be checked by observing whether the germ line cells are disturbed or
not. Pull out the capillary by moving the microscope stage away from the needle. It is essential to check whether or not
the needle tip is blocked. If the injection mixture does not flow out easily, press the right mouse button (Clean function)
until the blockage is removed.
Changing the capillary
If the capillary keeps being blocked, the needle has to be changed. Be very careful: the pressure can cause the needle
to shoot out like an arrow. To avoid this, always push the Menu button – "Option 0" – which will remove any pressure in
the needle.
Recovery
By gently touching the side of the worm with a picking rod, it will start to float in the oil. Remove the worm from the oil
and place it to a OP50 culture. After one hour, transfer the worm to a new OP50 culture plate to remove residual oil.
References
Brenner, S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71-94.
Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver et al., 1998 Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.
Mello, C. C., J. M. Kramer, D. Stinchcomb and V. AMBROS, 1991 Efficient gene transfer in C.elegans:
extrachromosomal maintenance and integration of transforming sequences. Embo J 10: 3959-3970.
Additional literature
Evans, Thomas C., Transformation and microinjection, WormBook, The C. elegans Research Community, WormBook,
http://www.wormbook.org.
Ordering information:
Article
FemtoJet express
TransferMan NK2
InjectMan NI2
PatchMan NP2
Microloader
Research Pipette
Order no. international
5248 000.017
5188 000.012
5181 000.017
5183 000.014
5242 956.003
3111 000.122
North America
920010521
920000011
920000029
920000037
930001007
022471902
A
U 012 WW 020/PDF/
02
06/KW
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