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

of chloroform.

 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.