DNA purification for transgene microinjection - GTTF will do this
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.