Co-CRISPR RNP Injection Mix Protocol

Notes:
 * 1) Resuspend target-gRNA and dpy-10-gRNA in IDT duplex buffer
 * 2) * Add 10ul duplex buffer to each to resuspend to 200uM gRNA
 * 3) ** Keep resuspended RNAs on ice to prevent degradation
 * 4) Resuspend dsDNA Ultramer oligo repair templates in water
 * 5) * Add 80ul RNAse-free water to resuspend to 50uM dsDNA oligo
 * 6) ** *If using plasmid repair template, be sure to phenol:chloroform purify it to remove RNAses before adding it to your mix
 * 7) Duplex 200uM target-gRNA and 200uM dpy-10-gRNA with 200uM tracrRNA
 * 8) * Add 2.25ul target gRNA, 0.25ul dpy-10 gRNA, and 2.5ul tracrRNA in RNAse-free microfuge tube
 * 9) * Incubate at 95°C for 5 minutes
 * 10) * Spin down briefly
 * 11) * Incubate at RT for 5 minutes
 * 12) ** ** The Zahler lab uses a 1:9 dpy-10-gRNA to target-gRNA ratio, and the Dernburg lab uses a 1:12 dpy-10-gRNA to target-gRNA ratio.
 * 13) Complex target-gRNA:dpy-10-gRNA:tracrRNA duplex with Cas9 protein
 * 14) * Add 5ul 40uM Cas9 protein (freshly thawed from -80°C) to 5ul duplexed RNA
 * 15) * Incubate at RT for 5 minutes
 * 16) Add target repair template
 * 17) * If using ssDNA Ultramer oligo repair template, add 1.3ul 50uM ssDNA target repair oligo, for ~6uM final concentration
 * 18) * If using plasmid repair template, add (phenol:chloroform purified) plasmid to final concentration of 10-30ng/ul
 * 19) Add dyp-10 dsDNA Ultramer oligo repair template
 * 20) Add 0.65ul 50uM dyp-10 ssDNA Ultramer oligo repair template to RNP mix for ~3uM final concentration
 * 21) Spin at 13,000RPM for 5 minutes
 * Always use barrier/filter tips to prevent contamination.
 * Always use RNAse-free microfuge tubes, tips, and water.
 * Work in an RNAse-free environment, wear full PPE, and change gloves often.
 * Always keep resuspended RNA on ice to prevent degradation.

**When screening, dumpy worms are usually dpy-10 homozygotes, which will need to be crossed out.