Viscous Crispr injection mix!

Does anyone else have difficulties with the viscosity of their injection mix clogging needles? I’ve been injecting plasmids for many years and can practically do it in my sleep! However, my Crispr mix (using similar concentrations
listed in the Seydoux protocol) clogs my otherwise ideal needles immediately. I even resuspended the Cas9 without glycerol. I’m getting incredibly low efficiency with my co-injection marker, since I basically insert the needle
and hit “clear” for a few seconds in hopes that something is going in. Thanks!


We used to have the same problem (particularly when we injected high concentrated Cas-9 mixes) until I adjusted properly the concentration of the HEPES buffer to the volume I was injecting, e.g. .5 microliter of HEPES 200mM (Ph 7.4 adjusted with 1500 mM KCL) in a 10 microliter injection mix.

On the other hand, you can try to greatly reduce the amount of CRISPR-Cas-9 as noted in:

I hope it helps!