Post-digest, my linearized plasmid looks to be the correct size by gel. However, post-transcription, the product is 0.5-1kb larger than expected (should be 4.4kb). Can I not rule out incomplete digestion even if the digest gel band was the expected size? What else could be going wrong here, and what can I do about it?
Albert Vill
Just to be clear, you are running your Cas9 RNA transcript on an agarose gel? If that's the case, make sure you're using an RNA size standard and not DNA. Single-stranded nucleic acids are less negatively charged than double-stranded nucleic acids of the same length. So, ssRNA will run slower and therefore appear larger than equal-length dsDNA.
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