Gene Targeting

Preparation of targeting constructs for electroporation

Targeting construct plasmids must be linearized prior to submission for electroporation. Standard methods of plasmid purification, such as CsCl gradient banding or commercial plasmid prep kits, are acceptable. Purified plasmid should be digested to completion with an appropriate restriction enzyme, extracted with phenol:chloroform (or phenol followed by chloroform, if preferred), ethanol precipitated, and resuspended at 1 µg/µl in TE or H2O. 25 µg of DNA is used per electroporation; we request that you submit 50 µg, in case re-electroporation is necessary.

Preparation of DNA from ES cells in 24-well plates

Many protocols are suitable for isolating DNA from ES cells of sufficient quality for PCR and a Southern blot. This is a straightforward, reliable protocol (from Manipulating the Mouse Embryo, Hogan et. al. 1994) which we can recommend, but investigators should use any protocol they feel comfortable with. Commercial genomic DNA prep kits have also been used with satisfactory results. Feedback about alternate protocols that may be easier or cheaper is welcomed.

From plates containing confluent ES cells:

  • aspirate medium, wash once with PBS, aspirate
  • add 200 µl lysis buffer; seal plate with parafilm, incubate at 55° a few hours to overnight
  • transfer lysate to microfuge tubes; add 100 µl saturated NaCl and shake vigorously
  • spin at 3000g for 15 minutes; transfer supernatant to new tube
  • add 2 volumes of 100% ethanol at room temperature; invert several times (precipitate should be visible)
  • spool out DNA (pipette tip, or heat-sealed end of pasteur pipette); rinse attached pellet in 70% ethanol, dry a few seconds, resuspend in 50 µl TE

Lysis Buffer:

  • 150 mM NaCl
  • 2 mM EDTA (pH 8)
  • 1% SDS
  • 20 mM TRIS-HCl (pH 8)
  • 50 μg/ml proteinase K

Saturated NaCl:

  • Add solid NaCl (with stirring) to 5M NaCl until it no longer dissolves.