Any standard procedure which will separate vector sequence from transgene sequence
Note: The Facility recommends the Qiagen Endofree plasmid kit (#12362); follow instructions carefully.
- Setup digest that cuts transgene sequences away from bacterial vector
- Make sure that restriction enzyme is not more that 10% of total digest volume
- Digest DNA to completion (may need to check aliquots on 1% agarose gel)
Gel separation of transgene from plasmid vector
- Prepare 1% LMP gel and run RE digested DNA
- Run gel far enough to clearly separate the transgene piece from any vector piece(s)
- Photograph and keep picture for documentation
- Excise transgene piece from gel
- Use Qiaquick Gel Extraction kit (#28704 for pieces under 5 kb and #20021 for larger pieces; make sure ETOH does not contaminate preparation)
- Other methods such as GeneClean also are acceptable
- Run an aliquot of cleaned DNA piece on gel for purity and estimated concentration
Additional cleanup for microinjection
The DNA transgene preparation for microinjection must be "clean" of all contaminants.
Otherwise, one runs the chance of destroying zygotes at microinjection.
DNA concentration is also a critical parameter.
An accurate estimate of the final DNA transgene preparation is required upon submission to the Facility.
- Concert Columns (BRL #11458-015): Follow the instructions in the package (Exception: steps 2,3,4 centrifuge at 1,000 x g)
- The DNA eluted in step 4 is dialyzed against microinjection buffer 2 times by pipetting a 50 µL aliquot onto a Millipore filter (#VCWP-013-00; pore size 0.10µm) floating in a 30 mm petri dish of microinjection buffer
- Leave undisturbed for 2 hours
- Collect the "dot" of DNA and wash spot on filter with an additional 50 µL of microinjection buffer
- Accurately quantitate transgene DNA on gel with markers of known concentration and submit this picture to the Facility with standards marked
- Final concentration of transgene DNA should be 10ng/µL microinjection buffer
10 mM Tris, pH 7.4, 0.25 mM EDTA, 10 mM NaCl in sterile water (available in aliquots from the Facility)
Protocol obtained from Mia Wallace, Director, Washington University Neuroscience Transgenic Mouse Facility