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Preparing samples for microinjection

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Why sample quality is important

Sample viscosity: When we are ready to use your sample, we transfer some of it to a glass microinjection needle. We then penetrate dechorionated embryos with the needle and use air pressure to force out some of the sample. The microinjection needle tip is several hundred times narrower than a standard pipette tip (see daiagram above). If your sample is viscous or contains any floating particles, it will not pass through the needle and your injection will fail. Please note that samples containing plasmids and other small nucleic acid molecules at our recommended concentrations should appear like water and should not be at all viscous.

Sample purity: We inject living embryos with a relatively large amount of fluid. The survival rate of the injected embryos is dependent upon the purity of the sample that is injected. If your sample is unusually toxic, your injection will fail. Please prepare nucleic acids using our recommended procedures below (unfortunately, we are not able offer this as a service). Ensure that your sample is in pure water and not buffer (e.g TE) as buffers are toxic to the embryo. Also, be aware that high concentrations of nucleic acid (above 1.0 ug/ul) in your sample will compromise embryo survival.

How to prepare plasmid DNA for microinjection

We recommend that plasmid DNA for embryo injection is prepared using the Qiagen mini-, midi- or maxiprep kit. If you use the Qiagen miniprep kit, you need to include the 'optional' PB buffer wash step. The OD260/280 ratio of your DNA sample should be 1.80-1.90. We acknowledge that other kits may generate DNA of the required quality for microinjection, but we will not recommend them until we have established that this is the case.

Adding helper plasmids to your microinjection sample

We can add helper plasmids to your sample to express the folllowing recombinases:

  • P-element transposase
  • PiggyBac transposase
  • PhiC31 integrase (for attP/attB recombination)

Please let us know on our microinjection request form if you want us to add helper plasmid to your sample. Also, please let us know in the 'Comments' section of the request form if you have already added helper plasmid to your sample. It is important for us to have this information as we review all requests for potential problems with stock selection, screening etc.

Recommended sample concentration and volume

Please provide us with at least 20ul of sample in a small (0.5 ml) Eppendorf tube. Please send your samples so that they are ready to inject. Our recommended concentrations are:

P-element transgenesis: Please send your P-element plasmid at approximately  1.0 ug/ul. We will added P-element helper plasmid. Final injection concentrations will be 0.8 ug/ul P element plasmid and 0.5 ug/ul helper plasmid.

PiggyBac transgenesis: Please send your PiggyBac plasmid at approximately 0.8 ug/ul. We will added PiggyBac helper plasmid. Final injection concentrations will be 0.6 for PiggyBac plasmid and 0.4 ug/ul helper plasmid.

phiC31 integrase-mediated transgenesis: Around 0.4ug/ul is the most common injection concentration. If you need us to add the integrase helper plasmid, please provide your sample at 0.5 ug/ul. Final injection concentrations will be 0.4 ug/ul for PhiC31 sample plasmid and 0.5 ug/ul PhiC31 integrase plasmid.

CRISPR: For CRISPR mutagenesis, we recomend that your guide plasmid concentrations is 0.1 ug/ul. For CRISPR homologous recombination, we recommend your sample contains 0.1ug/ul guide plasmid and 0.5 ug/ul donor plasmid. Please prepare the donor and guide plasmid mixture before sending your sample.

If you wish to differ from out recommended concentrations, that is fine, but please let us know your alternate concentrations for our records. Also, be aware that over 1.0 ug/ul of sample concentration can compromise embryo survival.

Contact Information

Tiny fly

E-mail contacts

Simon Collier (Fly Facility Manager): s.collier@gen.cam.ac.uk

General Fly Facility enquiries: flyadmin@gen.cam.ac.uk

Microinjection enquiries: flyinject@gen.cam.ac.uk

Fly food enquiries: flymedia@gen.cam.ac.uk

Fly stock requests: gh19@gen.cam.ac.uk

Address

Department of Genetics,
University of Cambridge, Downing Street,
Cambridge CB2 3EH,
United Kingdom

Phone: +44 (0)1223 765124
Fax: +44 (0)1223 333992

 

The Fly Facility webpages are maintained by Simon Collier. Please contact me ) if you have any problems using the site.