Protocols

Purification of DNA for Microinjection

  1. Recombinant plasmid should be purified by CsCl gradient (for details see Molecular Cloning: A laboratory manual, 2nd ed., Sambrook, et al., 1990). The insert is then released with the appropriate restriction enzymes. It is critical to remove all vector sequences, as such sequences can significantly alter the expression of transgenes (mechanism unclear).
  2. Separate the insert of interest from the vector on an agarose gel run in Tris/Acetate/EDTA (not Tris/ Borate/EDTA) buffer.
  3. Excise the gel slice containing the gene fragment of interest and electroelute the DNA fragment.
  4. Recover the fragment by ethanol precipitation, and then pass it through a Millipore column according to the manufacturer’s instructions.
  5. Ethanol precipitate the DNA and redissolve it in injection buffer (10 mM Tris/0.1 mM EDTA pH 7.5). This buffer must be prepared with distilled water to maintain zygote viability.
  6. Estimate the DNA concentration by comparing the ethidium bromide staining of a sample run on an agarose gel next to a standard of known concentration such as l DNA cut with Hind III.
  7. Submit the DNA sample in injection buffer.

Alternate Procedure

  1. After separating the fragment on agarose gel run in TAE, excise the fragment of interest from the gel.
  2. Process the gel fragment through Geneclean II kit (Bio 101, Inc.) or Qiaex gel extraction kit (Qiagen, Inc.) according to the manufacturer’s instructions.
  3. Ethanol-precipitate the recovered DNA and resuspend in injection buffer
  4. Pass through a Millipore Column (Millipore).
  5. Ethanol precipitate the recovered DNA and resuspend in injection buffer (10 mM Tris/0.1 mM EDTA, pH 7.5 prepared with distilled water).
  6. Estimate the DNA concentration by comparing the ethidium bromide staining of a sample run on an agarose gel next to a standard of known concentration.
  7. Submit the DNA sample in injection buffer.

Notes

  • For ethanol precipitation of a sample, add 1/10 volume of 3 M NaAc and 2-2.5 vol. ethanol.
  • Use 1:100 dilution of EtBr (5 mg/ml) for gel staining. Visualize the DNA band with long-wave UV light to avoid DNA damage.
  • It is strongly recommended that powder-free gloves be used when handling the DNA preparation. Powdered gloves are most often associated with the presence of particulate matter that gets lodged at the tip of the injection needle and renders it useless.