Currently we use plasmid purification kits. These kits come in different sizes. Follow the kit instructions. Always pre-heat TE to 65°C before final purification step. In the old days we used the manual procedure using Solution 1, 2 & 3. But no more!
Preparing plasmid DNA for microinjection in pro-nuclei to make transgenic mice (Cyagen)
Prepare plasmid DNA with any of the following kits: NucleoBond PC20 Kit (Clontech: Cat# 740571); Qiagen EndoFree Plasmid Kit (Cat# 12362); or Qiagen Plasmid Maxi Kit (Cat# 12162).
Separate the insert from the vector backbone by restriction digestion of 20 μg of plasmid DNA.
Electrophorese the sample on 0.8% agarose gel using 1x TAE buffer containing 0.1 - 0.2 μg/mL ethidium bromide.
Excise the fragment under long-wave UV light using a clean scalpel (short-wave UV light is not recommended because it may break DNA).
Purify the fragment as follows:
a. For DNA fragment smaller than 10 Kb, use Qiagen Qiaquick Gel Extraction Kit (Cat# 28704). Elute DNA in 40 μL plasmid injection buffer during the final step.
b. For DNA fragment from 10 - 40 Kb, use Qiagen QIAEX II Gel Extraction Kit (Cat# 20021). Elute DNA in 40 μL plasmid injection buffer during the final step.
Measure DNA concentration by a spectrophotometer such as NanoDrop.
Check 100 ng DNA on an agarose gel by comparing an aliquot of the DNA sample with size standard of known concentration (e.g., Gibco High DNA Mass Ladder: Cat# 10496-016). Store DNA at 4°C.
Submit at least 50 μL of DNA with a concentration of at least 100 ng/μL in plasmid injection buffer along with the picture of the gel to us for microinjection. It is okay to ship the DNA at room temperature.
Buffer:
Plasmid injection buffer:
- 7.5 mM Tris
- 0.2 mM EDTA
To make 40 mL plasmid injection buffer:
- 1 M Tris-HCL, pH 7.4 (can be purchased from Sigma, Cat# T2663): 300 μL
- 0.5 M EDTA (Sigma, E7889): 16 μL
- Add dH2O (high quality) to 40 mL