Eppendorf 012: Microinjection of plasmid DNA or double stran Manual de usuario descargar pdf (Pagina 3)

Description

Programmable microinjector with external

pressure supply

Proportional 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

AU 012 WW 020/PDF/0206/KW

●

eppendorf

is a registered trademark

Your local distributor: www.eppendorf.com/worldwide Application · Support: E-Mail: support@eppendorf.com

Eppendorf AG · Germany · Tel. +49 40 538 01-0 · E-Mail: eppendorf@eppendorf.com

Eppendorf North America, Inc. · USA . Tel. +1 516 334 7500 · Toll free phone 800 645 3050 · E-Mail: info@eppendorf.com

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