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Mouse Transgenics Protocols

Purification for Microinjection Protocol

One of the most critical parameters in the success of transgenic experiments is the quality of the DNA. DNA provided for microinjection must be free of biological and chemical contaminants and particulate material. Linearized DNA constructs, for microinjection, can either be provided by the client in purified form or purified by the transgenic core. It is important that clients providing DNA to the core facility follow the procedure outlined below.

Note well: To decrease the particulate matter in the microinjection DNA sample, it is important to use embryo tested water (Sigma Aldrich W1503) for all prepared solutions, to filter all solutions through a 0.2 µm filter and to use new plastic containers and tubes for all solutions.

  1. The gene of interest should be cloned into a plasmid vector and the plasmid purified by CsCl gradient or using the Qiagen plasmid purification system.
  2. Excise the DNA fragment of interest from the plasmid vector by restriction enzyme digestion.
  3. Analyze the restriction digest by gel electrophoresis using ultrapure agarose and excise the band containing the microinjection DNA.
  4. Purify the DNA from the gel using the Qiagen QIAquick kit,the Stratagene Nucleospin kit or the S&S Elutip system.
  5. Ethanol precipitate the DNA and wash the pellet twice in 70% ethanol.
  6. Resuspend the DNA in a small quantity of filtered injection buffer (5 mM Tris, pH 7.4, 0.1 mM EDTA) prepared in embryo tested water.
  7. An aliquot of this preparation should be analyzed by gel electrophoresis to confirm that the preparation contains a single DNA fragment of the correct size. This photo must be submitted with the DNA sample to the VARI transgenic core.
  8. A total of 5 µg of linearized fragment is required by the facility. Please clearly mark the DNA concentration on the tube and on the application form.
  9. Ship the DNA on dry ice by overnight delivery service.
  10. If you experience any problems during the purification of the DNA fragment or if you have any questions, please contact the VARI Transgenic Core for advice.

Transgenics DNA Isolation from Mouse Tissue Protocol

  1. Excise ~0.5 cm of the mouse tail tip and place in a 1.5ml eppendorf tube.
  2. Add 400 µl of tissue digestion buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.5% SDS) containing 50 µg/ml Proteinase K (Roche Molecular)
  3. Incubate overnight at 55°C with gentle shaking.
  4. Allow the tubes to cool to room temperature and flash spin to remove the solutions from the tube lid.
  5. Add 400 µl of phenol/chloroform solution (1:1).
  6. Close the tubes and mix vigorously by shaking or inverting the tubes (do not vortex).
  7. Spin in a microfuge at maximum speed for 5 minutes.
  8. Transfer the top aqueous phase to a new 1.5 ml eppendorf tube. Use a large orifice pipetman tip to transfer.
  9. Precipitate the DNA by adding 2 volumes of absolute ethanol and shake vigorously until the precipitate is visible.
  10. Spin in a microfuge at maximum speed for 4 minutes. Decant the supernatant.
  11. Wash the pellet with 200 µl of 70% ethanol.
  12. Spin in a microfuge at maximum speed for 1 minute. Decant all of the supernatant.
  13. Air dry the tubes.
  14. Resuspend the DNA pellet in 200 µl TE buffer (10 mM Tris, 1 mM EDTA). Resuspension of the DNA can be aided by incubating the tubes at 37°C for several hours.