Protocols

Preparation of DNA for CRISPR Knock-In Projects (for DNAs larger than 500 bp)

1. Prepare 30-50 µg of supercoiled plasmid DNA. We REQUIRE that you use an endotoxin-free plasmid preparation protocol that results in endotoxin-free, clean plasmid DNA. The EndoFree Qiagen kit is suitable for this purpose.
2. Run the DNA on a gel to verify that it is supercoiled. Compare to molecular weight markers of the appropriate size. Take a clear picture of this gel and bring the picture with your DNA to the GEMF.

The facility will further purify the fragment for injection.

Preparation of DNA for Electroporation into ES Cells

1. Prepare at least 30 µg of the targeting vector plasmid. We recommend the Qiagen EndoFree Plasmid Maxi kit. Alternatively, one can use standard plasmid protocols followed by CsCl banding
2. Linearize the targeting vector by digesting with a unique enzyme
3. After digestion, check for complete linearization by running a small fraction on a mini-gel
4. Precipitate DNA with two volumes of ethanol and 0.3 volume of 7.5 M ammonium acetate
5. Wash pellet 2X with clean 70% ethanol
6. Resuspend DNA in 0.1X TE* at concentration of 1.0 mg/ml. A total of 25 micrograms at a concentration of 1 μg/μl of linearized DNA is needed for electroporation.

Note:

• It is very important to precipitate DNA with ammonium acetate rather than the commonly used sodium acetate
• It is equally important to resuspend the DNA pellet in 0.1X TE according to the protocol above and not water or PBS
• Not adhering to either of the above will greatly affect transformation efficiency

Preparation of DNA for Pronuclear Injection

1. Prepare 30-50 µg of DNA  using an endotoxin-free process. We REQUIRE that you use an endotoxin-free plasmid preparation protocol . The EndoFree Qiagen kit is suitable for this purpose.
2. Digest enough transgene construct to release 20 µg of fragment to be injected. (The fragment to be injected has to be free of all vector sequences.)
   
3. After digestion, check for complete digestion by running a dilute portion (less than 50 ng) of the DNA on a gel. Compare to a lane of uncut plasmid and a lane where the digestion is overloaded (greater than 150 ng) (to check for additional unwanted products). Take a clear picture of this gel and bring the picture with your DNA to the GEMF.

The facility will further purify the fragment for injection.

Please contact the facility director for special preparation procedures for large DNAs (YACs, BACs, etc.)