Preparation of DNA for pronuclear injection
- Cut out all plasmid sequences from the construct. Use a restriction enzyme leaving cohesive ends (NO “blunt ends”). In the digest you should have at least 30 micrograms of the plasmid insert to be injected.
- Agarose gel electrophoresis: use low current to better separate the fragments. To avoid contamination, clean the electrophoresis equipment thoroughly and use clean reagents.
- Take a picture of the gel to be taken to the GM mouse unit with the DNA. Cut out the insert fragment from the gel and extract the DNA (for example Qiagen QIAEX or QIAquick Gel Extraction –kit).
- Dry the DNA pellet and resuspend in filtered buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5). Check concentration and integrity by agarose gel electrophoresis with DNA-standards. Take a picture and take it to the GM mouse unit with the DNA. Adjust concentration to 5 ng/microliter (in 10 mM Tris, 0.1 mM EDTA, pH7.5), the total volume should be min 200 microliters. This concentration is on the borderline of being toxic to the oocytes, but gives the best insertion frequency. Therefore, careful adjustment of the concentration is important.
- Centrifuge the DNA in a microcentrifuge full speed (13 000 rpm) for 10 min. Transfer 90% to a clean sterile tube. Avoid repeated freezing and thawing and long-term storage.
Note ! You can get the filtered buffer from the GM mouse unit (10 mM Tris, 0.1 mM EDTA, pH7.5)