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Sapphire Unicornover 4 years ago

I've used NEB Cas9 in zebrafish.

I make an injection mix in the following way:

0.3 ul 0.5% phenol red dye

1 ul cas9 buffer (see recipe below, to stop protein from crashing out of solution)

1 ul sgRNA(s) @ 200-500ng/ul

1 ul Cas9 protein (highest concentration available from NEB, IDT, etc)

buffer is 200 mM KCl, 10 mM MgCl2, 20 mM Tris pH 8

Add ingredients to the mix in the order above to avoid adding sgRNAs directly to Cas9 protein, that can make the protein crash out and clog your needle.

Inject as much as you can before toxicity happens, usually 1.5 nL.

Some more details in the Supp Protocol of our 2014 paper here - http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098186

Hope this helps - good luck!

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