Multichannel Systems Roboinject Manual - Manual (Page 54)

Roboinject Manual
50
4.3 Preparations for Injection
Generally, you can use cDNA or mRNA concentrations and injection volumes as described in
the literature or as applicable for your experiment. In the following, typical cDNA or mRNA
concentrations and recommended treatment of the sample are described. Please follow this
advice if you are new in operating the Roboinject or if you have observed trouble with the
injection process.
You will also need the provided stereo microscope and alignment device (with crosshairs)
for the alignment of the injection needle. Please read chapter "Alignment".
Note: The cDNA or mRNA sample has to be very clean and pure to obtain good results.
Any particles, dirt, dust, and so on could clog the needle. Please check the sample for protein
contaminations, cellular debris, residues of the extraction kit, or a high salt content.
Checking the oocyte quality
You need oocytes plated in standard 96 well plates. You should only use oocytes of a very high
quality and of uniform size. Make sure that all oocytes are positioned with the animal pole (dark
side) up, when you want to inject cDNA into the nucleus. The oocytes will have adhered to the
well bottom after about 2 to 3 hours. Do not use the cells before. Best results have been obtained
if oocytes have been incubated over night before use. See also "Preparation of Xenopus Oocytes"
in the Appendix.
cDNA / mRNA Concentration and storage
 Depending on the efficiency of protein expression, about 10 to 50 ng of cDNA (about 250
to 2500 ng of mRNA) is sufficient to maximally express the desired receptor in all 96 oocytes.
Much lower amounts might be appropriate if you need a reduced expression level,
for example for electrophysiological recording of ion channels.
 A typical cDNA sample has a concentration of 10 to 50 ng/μl, a typical mRNA sample
has a concentration of about 50 to 500 ng/μl.
 DNA or RNA should be stored in sterile, nuclease-free 0.5 ml or 1.5 ml Eppendorf tubes
(or similar reaction tubes) at -80 °C.
 Highly concentrated stock solutions should be diluted with nuclease-free water before use.
Do not use saline solutions because salt crystals may form during injection and may cause
the needle to clog.