Guidelines for preparing CRISPR/Cas9-expressing DNA plasmids for CRISPR injections (10/21/14)


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Protocol
Record History
Added on July 16, 2014 at 5:09 PM by Mortlock, Douglas
Modified on October 21, 2014 at 7:14 PM by Mortlock, Douglas
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VGER: Protocols Master

To purify CRISPR/Cas9-expressing DNA plasmids, such as modified PX330 that express customized guide RNAs and Cas9 mRNA, for CRISPR injections, please follow these general guidelines. 

Perform a standard Qiagen kit midi prep or maxi prep.  Qiagen-purified DNA has historically performed well in terms of embryo survival, so we prefer it.  Midi- or Maxi-preps are required, as minipreps may not provide quite enough plasmid DNA for multiple injection days.  

To remove contaminants that can negatively affect embryo survival, it is very important to carefully rinse the DNA pellet with 70% ethanol at the precipitation stage. Note, the Qiagen Hi-Speed maxiprep kit has an unusual ethanol rinse step that in our experience may not remove all contaminants.  If using this kit, simply re-precipitate the DNA using standard ethanol/sodium acetate and centrifugation, followed by 70% ethanol rinse.  

Following 70% ethanol rinse, carefully remove all traces of ethanol from the pellet and allow to air-dry for 5 minutes.  Resuspend the DNA in 10 mM Tris pH 7.4 or Lo-TE (10 mM Tris pH 7.4, 0.1 mM EDTA).  Provide at least 20 µg uncut, circular DNA to the core at a minimum concentration of 100 ng/µl.    Clean DNA should have a 260/280 ratio of approximately 1.85 and a 260/230 ratio of at least 2.