Multichannel Systems Roboinject Manual - Manual (Page 53)

Injection
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Xenopus oocytes
The impalement and injection distances for cDNA are preset to 300 μm and 550 μm, respectively.
(from the bottom of the well = 0 value).
These distances have been shown as the optimum values for DNA injection. The overall expression
rate you can achieve by this method is 80 percent of the oocytes. You can define your own
impalement and injection depth, but MCS recommends using the default values in order to
achieve the best expression rate.
You may wonder why the injection needle is placed so deeply for cDNA injections while the
nucleus is known to be located near the animal pole. As it seems, the nucleus is pressed down
by the injection needle until the pressure is sufficient for the needle to penetrate the membrane
of the nucleus.
Messenger RNA is injected into the cytoplasm. Generally, it is not important where in the
cytoplasm the RNA is released. The default impalement and injection distances for mRNA
are 650 μm and 550 μm, respectively. These are safe values even for smaller oocytes.
Running a test for nuclear injection with a dye
To demonstrate the precision of the injection and to practice the general handling, you may
consider running a test injection with the dye "Trypan Blue", which stains proteins unspecifically,
before you start to inject your constructs. Injected oocytes are then torn open with forceps, thus
releasing the complete, stained nucleus.
1. Dissolve Trypan Blue in distilled water. The solution should appear bright blue.
2. Remove any particles with a 0.22 μm syringe filter or something similar.
Continue with the next chapter "Preparations for Injection". You can use this dye solution
to mimic cDNA. If you have successfully performed the test or if you prefer to start your first
experiment under real conditions, you can right away start to use your cDNA or mRNA.
You may consider the test injection again if you observe any troubles.
The following pictures show injected oocytes. In the left picture, the penetration site of the
needle is clearly visible in the middle of the animal pole. The right picture shows an opened
oocyte. Only the nucleus is stained, showing that the nucleic membrane is still intact and
therefore impermeable for the dye.