Preparation of DNA for Electroporation of ES Cells
The following protocol is taken directly from Manipulating the Mouse Embryo, 3rd Edition, Nagy et al.
Please provide at least 30ul of purified DNA construct at a concentration of 1µg/µl.
The DNA must be high quality and free of all contaminating chemicals.
- Purify the vector DNA using a Qiagen column (Qiagen 12143 or 12362) or by CsCl centrifugation.
- Linearize DNA.
- Run a sample of cut DNA on mini-gel to check for complete linearization.
- Extract the DNA with phenol : chloroform : isoamylalcohol (25:24:1) (Invitrogen #15593-031) OR with an equal volume of 1:1 phenol : chloroform, then equal volume of chloroform.
- Precipitate with two volumes of absolute ethanol on ice, spin down, can be stored at -20 C from this point.
- Wash two-three times in 0.5-1 ml of 70% ethanol
- Drain off as much 70% ethanol as possible and allow the remainder to evaporate in a sterile laminar flow hood, leaving the lid of the tube open for 30-60 min (optional)
- Re-suspend in ultrapure sterile water, take an aliquot for measurements (can freeze the rest at – 20C for long term storage)
- Quantify OD260nm:280nm. If possible, use a NanoDrop to quantify and assess the purity of the final prep. Please, refer to the NanoDrop Nucleic Acid Purity ratios.