Protocols

Targeting Vector Linearization

Prepare approximately 200-250 µg of linearized DNA

1. Perform the appropriate restriction enzyme digest in a volume of 0.5 µg/µl digest.
2. Monitor complete digestion by running an aliquot on a gel.
3. Phenol/Chloroform extract two times (use at least 1 volume of Phenol/Chloroform).
4. Chloroform extract twice (use at least 1 colume of chloroform).
5. Ethanol precipitate the DNA.
6. Wash extensively in 70% Ethanol.
7. Spin and rewash a couple of times.
8. Air dry the DNA pellet.
9. Resuspend in ES cell grade water or TE made with ES cell grade water at ~1 µg/µl.
10. Label tube with Principal Investigator's name, DNA density, Vtotal and date.

Notes:

• 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 water, therefore, extreme care should be taken with the extractions and with precipitating 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.
• Steriliy is another important issue, therefore, especially after the extractions, manipulations should be made 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 resuspended, should be stored at -20°C.