Problem	Possible Cause	Solution
Cas9 mRNA degraded	RNase might have been introduced into the system.	One must be mindful that RNase is ubiquitous in a laboratory environment and exercise vigilance by decontaminating countertop and all glassware and plastic wares that are being used to support Cas9 mRNA synthesis and use RNase free tips and vials. Also avoid talking while working with RNA samples.
No or low recovery of the sgRNA from column purification	The MEGAclear Kit (Part Number AM1908, Life Technologies) is a glass filter-based system for purification of single stranded RNA transcript and suitable for RNA transcripts greater than 100 nt. The sgRNA in a CRISPR/Cas9 experiment is often 120 nt in size and may not be adequately retained in the column.	Consider using phenol:chloroform extraction and alcohol precipitation to purify the sgRNA preparation instead. Or use alcohol precipitation directly.
Microinjection mixture clogging the needle	This may be caused by particulate matter carryovers from column purification or a high concentration of the donor DNA.	It can be resolved by centrifuging the microinjection mixture at 20,000 × g for 15 minutes and using the supernatant for microinjection.
No or low live birth for manipulated embryos	Embryos compromised in the process	Whenever available, transfer unmanipulated embryos as a control to determine whether media, reagents or environmental conditions (excessive noise, construction, etc.) may be the problem.
No targeting	The guide may not have worked	Choose another guide or carry more than one guide in parallel if possible to maximize the chance of successful delivery of the model at the end of the process. Also it may help to evaluate the guides in vitro in a cell line or injecting into mouse embryos and screen the guides before choosing a guide for a full-scale experiment.
Poor recovery of the embryos from the electroporation cuvette	The embryos may have adhered to the sides of the cuvette	Be sure to use the plastic transfer pipette that comes with the cuvette to transfer embryos. Also, the embryos can be recovered by an additional round of rinsing of the cuvette.