In Vitro Transcription (IVT) Cas9 or Cas9-mSA mRNA for microinjection

Cas9 is cloned into the pCS2 IVT plasmid containing an SP6 promoter.

Linearize plasmid using NotI-HF.

Run 5μl on gel to check complete linearization. Purify rest by ethanol precipitation. Resuspend in RNase-free water. Quantify using Nanodrop.

Cas9 is then in vitro transcribed using the Ambion mMessage mMachine SP6 Kit according to the manual.

NCAP                                        20μl

10x buffer                                  4μl

2μg linearized Cas9-pCS2            x μl

ddH2O                                  to 40μl

total                                                40μl

Incubate >4 hours 37°C.

Add 2μl Turbo DNase and incubate 15 min 37°C.

Cas9 mRNA purification: 

For RNA cleanup use RNeasy Mini Kit (Qiagen, catalogue number 74106). Elute in 30-40μl RNase-free water. 

Quantify using Nanodrop, yield should be between 600-1200ng/μl.

Run 2μl on gel to check integrity. 

Aliquot Cas9 mRNA and store at -80°C.