I have a plasmid (~9.3kb) with a small 20 nt insert, which I created using the Q5 Site-Directed Mutagenesis Kit via PCR. Before transformation, I obtained a clear band at 9.3 kb.

I picked up a single colony into competent E. coli and let it grow overnight. The next day, I performed a MiniPrep to extract the DNA and quantified the concentration using NanoDrop. The DNA concentration was around 200 ng/µl, with an A260/A280 ratio between 1.9 and 2.0.

I then analyzed the plasmid on a 1% agarose gel after linearizing it with a restriction enzyme (RE) for 2 hours at room temperature. I expected to see a clear band around 9.3 kb.

However, I observed multiple bands in the gel, which has left me confused about the results. Do you have any suggestions or ideas about this data?

Please share your thoughts!

Thank you, and I wish you success in your life science research.