This protocol may be used to linearize DNA prior to gene targeting.


Category
Protocol
Record History
Added on June 29, 2010 at 11:05 AM by Lindner, Jill
Modified on July 2, 2010 at 10:47 AM by Lindner, Jill
Shared with (contributions)
TMESCSR: Protocols Master

Reagents, supplies and equipment:

  • appropriate restriction enzyme
  • phenol
  • chloroform
  • 70 % ethanol
  • ES cell grade H2O or TE

Procedure: Prepare approximately 200 - 250 µg of linearized DNA by either Qiagen or CsCl banding.

  1. Perform the appropriate restriction enzyme digest in a volume = 0.5 µg/ml digest.
  2. Monitor complete digestion by running an aliquot on a gel.
  3. Phenol/chloroform Extract 2 times.  (Use AT LEAST 1 volume of phenol/chloroform.)
  4. Chloroform extract twice.  (Use AT LEAST 1 volume of chloroform.)
  5. Ethanol precipitate the DNA.
  6. Wash extensively in 70% ethanol.
  7. Spin and rewash a couple of times. (Wash 3 times total.)
  8. Air-dry the DNA pellet.
  9. Re-suspend in ES cell grade H2O or TE made with ES Cell grade H2O at ~1 µg/ml.
  10. Label tube with principal investigator's name, DNA concentration, Vtotal, and date.

Notes:

  1. A large quantity of DNA is required, especially if multiple experiments become necessary.
  2. 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.
  3. Sterility is another important issue; therefore, especially after the extractions, manipulations should be performed in a tissue culture hood with sterile reagents, tubes etc. 
  4. Linearized DNA is much more efficient for gene targeting, so take the time to verify complete digestion BEFORE proceeding to clean up the DNA.
  5. Linearized DNA is more susceptible to exonuclease degradation therefore once re-suspended it should be stored at -20°C.