Mouse Genetics Facility

Helpful Information

Preparation of transgene DNA constructs for pronuclear injection
The Facility requires a minimum of 1–2 µg DNA construct fragment in
a volume of 20 µl Tris-only buffer.

1. Digest recombinant DNA plasmids with the appropriate restriction enzyme(s) to release transgene DNA construct fragments from vector backbones. (The Facility recommends running a pilot digestion to optimize conditions for a complete digestion, then using a large amount for the actual digestion.)
   
   
2. Run a small aliquot on an agarose gel to verify complete digestion and DNA integrity.
   
   
3. Load the entire digest into either a standard or low-melt agarose gel in TAE buffer to separate the DNA fragments from others (see table below).
   
   
4. Use a hand-held long-wavelength UV light to visualize and excise the desired DNA fragments. (Note: Short-wavelength UV light damages the ends of DNA fragments.
   
   
5. We recommend using QIAEX II Gel Extraction Kit (QIAGEN) to extract and purify DNA fragments (up to 50 kb). Follow the manufacturer’s instructions. (Note: Use only 10 mM Tris-Cl buffer (included in the kit) as an elution buffer. TE buffer should never be used.)
   
   
6. Run a small aliquot on an agarose gel after elution to verify DNA integrity. The gel image can be submitted to the Facility along with the DNA fragments
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7. Measure the concentration of the DNA fragments and evaluate the purity of the preparation by spectrophotometry (or suitable alternative).
   
   
8. If delivering the DNA fragments to the Facility immediately, wrap the DNA tube with Parafilm and store at 4°C. Otherwise, store at -20°C.
   Separation range of DNA in TAE gels with different concentrations of agarose