PREPARATION OF DNA FOR ELECTROPORATION OF ES CELLS*
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.
1. Purify the vector DNA using a Qiagen column (Qiagen 12143 or 12362) or by CsCl centrifugation.
2. Linearize DNA.
3. Run a sample of cut DNA on mini-gel to check for complete linearization.
4. 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
5. Precipitate with two volumes of absolute ethanol on ice, spin down, can be stored at
-20 C from this point.
6. Wash two-three times in 0.5-1 ml of 70% ethanol
7. 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)
8. Re-suspend in ultrapure sterile water, take an aliquot for measurements (can freeze
the rest at – 20C for long term storage)
9. 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.
*taken from The University of Washington: Transgenic Resource Program website.