Reagents, supplies and
- appropriate restriction enzyme
- 70 % ethanol
- ES cell grade H2O or TE
Prepare approximately 200 - 250 µg of linearized DNA by either
Qiagen or CsCl banding.
- Perform the appropriate restriction enzyme digest in a volume =
0.5 µg/ml digest.
- Monitor complete digestion by running an aliquot on a gel.
- Phenol/chloroform Extract 2 times. (Use AT LEAST 1 volume
- Chloroform extract twice. (Use AT LEAST 1 volume of
- Ethanol precipitate the DNA.
- Wash extensively in 70% ethanol.
- Spin and rewash a couple of times. (Wash 3 times total.)
- Air-dry the DNA pellet.
- Re-suspend in ES cell grade H2O or TE made with ES
Cell grade H2O at ~1 µg/ml.
- Label tube with principal investigator's name, DNA
concentration, Vtotal, and date.
- A large quantity of DNA is required, especially if multiple
experiments become necessary.
- ES cells are extremely sensitive to residual organic compounds
and endotoxins present in typical laboratory reagents and
H2O, therefore extreme care should be taken with the
extractions, precipitation, and washing the DNA. Extreme care
goes into the preparation of ES cell media and similar quality
reagents should be used to prepare the DNA solution.
- Sterility is another important issue; therefore, especially
after the extractions, manipulations should be performed in a
tissue culture hood with sterile reagents, tubes etc.
- Linearized DNA is much more efficient for gene targeting, so
take the time to verify complete digestion BEFORE proceeding to
clean up the DNA.
- Linearized DNA is more susceptible to exonuclease degradation
therefore once re-suspended it should be stored at -20°C.