Transgene DNA Purification Protocol
DNA purification for transgene microinjection – GTTF will do this for you.
1. Recombinant plasmid DNA should be purified by CsCl gradient or Qiagen column. The insert must then be separated from the vector by restriction digestion, since vector sequences can significantly alter the expression of transgenes (mechanism unclear). Please provide us with the restriction digestion tube, containing 10 micrograms of plasmid DNA, buffer and enzymes. This should be after incubation at the proper temperature, and you should check with a gel that this digestion has gone to completion.
50 microliters of volume is good, but we can handle more. Please tell us if we must use special gel conditions or a very long run to separate your band from others.
2. Provide a gel photograph of the same DNA preparation and restriction digestion, with the correct transgene band marked. We will use this to ensure that we isolate the proper band for your project. We also will need a map of your transgene and excision strategy, with the expected sizes of all expected bands.
If your laboratory is unable to follow this procedure, please discuss any changes in protocol with us prior to submitting the construct for microinjection.