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UCSD Transgenic Mouse and Gene Targeting Core
 

Summary: UCSD Transgenic Mouse and
Gene Targeting Core
9500 Gilman Drive, MC 0687
La Jolla, CA 92093-0687
(858) 534-3178
Current as of 3/01 1
TRANSGENIC MICE: METHOD FOR PREPARING DNA
FOR MICROINJECTION
(Courtesy of Rosenfeld lab members UCSD)
1. Grow your plasmid in a bacterial strain like XL-1 Blue (Stratagene) that has no
endoA activity and does not produce a lot of carbohydrates. Grow a 250-500 ml
culture from a fresh transformation.
2. Make a Qiagen-500 DNA preparation according to the manufacturer's instructions.
Resuspend DNA in 500 ul TE.
3. Further purify the DNA obtained from the Qiagen preparation over a CsCl gradient.
Add 50 ul EtBr (10mg/ml) to DNA sample. Add sample to 4.5 ml CsCl solution (0.9
mg/ml). Place sample in sealing tube, heat seal, and spin in ultracentrifuge at 78K for
3-4 hours. Remove band, add 1 volume of TE, and extract 4-5 times with H2O
saturated butanol, to remove ALL EtBr (this is critical). Precipitate DNA and
resuspend in 400 ul TE.

  

Source: Abagyan, Ruben - School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego

 

Collections: Biology and Medicine