Preparation OF CRISPR/Cas9 mRNA For Microinjection
The following protocol was adapted from the Transgenic Core at the University of Michigan.
- in vitro transcribed mRNA
- Oligonucleotide resuspended in RNAse free water
- DNA donor plasmid prepared with an endotoxin free plasmid kit
- 0.02 um Anotop 10 Syringe Filters (Whatman Cat. No. 6809-1002)
- Do not filter nucleic acid solutions through the Anotop filters, the 0.02 um pores
- will trap nucleic acids..
- 1 M Tris-HCl, pH 7.4 (Sigma Cat. no. T2663)
- 0.5 M EDTA (Sigma Cat. no. E7889)
- Nuclease Free Water (Sigma Cat. no. W4502)
RNase-Free Microinjection Buffer:
*All ingredients and supplies, should be RNase-free.
RNase Free Microinjection Buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA)
For 10 ml For 20 ml
Mix Together Mix Together
9.9 ml water 19.8 ml water
0.1 ml Tris-HCl, pH7.4 0.2 ml Tris-HCl, pH 7.4
0.005 ml EDTA 0.010 ml EDTA
Wash 0.02 um filter with 1 ml buffer – discard wash
Filter remaining buffer through filter for use as dialysis buffer or for use as microinjection buffer.
mRNA and sgRNA are transcribed in vitro with kits and procedures as described (Geurts et al, 2009, Wefers et al., 2013, Yang et al., 2013).
To avoid clogging microinjection needles, wash buffers and elution buffers used in mRNA and sgRNA purification should be pre-filtered through 0.02 um filters. Do not pass nucleic acid solutions through the filters as the pore size on the filters is small enough to trap nucleic acids.
Plasmid DNA donors should be purified with endotoxin free kits. Pre-filter all buffers through 0.02 um filters. Do not pass nucleic acid solutions through the filters.
Mix together nucleic acids in 0.02 um filtered RNAse-Free Microinjection Buffer at the desired concentrations. Prepare several aliquots of 50ul in 1.5ml microtubes. Store at -80°C.
100ng/ul Cas9 mRNA + 50ng/ul gRNA (+ 200ng/ul oligo)