DNA Purification for transgenic microinjection

Take extreme care to minimize particulate matter or other contaminants in the linearized gene fragment solution. Prepare at least 100ul of a 5ng/ul solution=> 0.5ug total.
  1. After cloning your gene construct purify by cesium chloride gradient or Qiagen column.

  2. Restriction digest the vector separating the fragment for injection.

  3. Run the digested DNA on an agarose gel and stain with ethidium bromide.

  4. Visualize with longwave uv light to avoid degradation and excise the band containing the gene construct.

  5. To free the DNA from the agarose either electroelute or use a Qiaex gel extraction kit sold by Qiagen.

  6. Add 1/10 volume of 3M Acetate and ethanol precipitate in 2-2.5 volumes of absolute ethanol. Resuspend in elutip or NACs buffer.
  7. Pass the DNA through either an elutip-D mini-column sold by Schleicher and Schuell or pass through a NACS column sold by Gibco BRL

  8. Ethanol precipitate and wash well with 70% ethanol. Be careful to evaporate the ethanol completely and resuspend your DNA in filter sterile TE (10mM tris, 0.1mM EDTA, pH 8) at 3 to 5ng/ul. Andrew will need at least 75 to 100 ul of this DNA solution.

  9. Spin in a microfuge for 15 minutes at 14,000 and then pipette off the upper 100ul of DNA for injections.