FAQ: The sgRNA is not running as a single band on the gel – why is this happening?

We recommend running the RNAs on a TBE-Urea (denaturing) gel under denaturing conditions.  The sgRNA is highly structured and if not fully denatured may run as multiple bands on a gel and may not run at the correct size as compared to a ssRNA ladder such as the Low Range ssRNA Ladder (NEB# N0364). If smearing is present, this may indicate contamination with an RNase.  We recommend wearing gloves and using nuclease-free tubes and filter tips