To improve plasmid quality for in vitro transcription, remove genomic DNA by using RNase treatment and better extraction kit. If the circular plasmid appears larger than expected, it might be due to aggregation or handling issues, so use fresh buffer and handle gently. If the circular plasmid is nicked or relaxed, use topoisomerase to fix it.

Use a plasmid prep kit that removes genomic DNA, handle samples carefully to prevent breakage, and ensure optimal bacterial growth conditions. Always check the plasmid quality and quantity before using it for transcription.

You are correct that supercoil plasmid should run faster than linear plasmid, although open circle plasmid (nicked) runs with similar mobility as linear.

I'm not sure how much bigger your uncut plasmid is running relative to the linearized, but it may be that you have multimers (dimers, trimers etc). Or your plasmid prep may be comprised mostly of nicked open circle.

What host strain are you purifying the plasmids from ? Sometimes a change in host might give you a better plasmid prep.

Thank you again for your help! I hope my experience can help. did it by improving the quality of the plasmids. I was using the phenol-chloroform method as described in the manual to purify it after linearization, which has a process of freezing the purified plasmid with NaAC and Ethanol for precipitation. It was time-consuming and harmful to my plasmid. Because the plasmid template could somehow inactivate during the freeze-thaw cycle. I finally used the purification kit, which is easier and allows me to use fresh plasmid every time. I am kind of sure thatan this freeze-thaw cycle damaged the plasmid (or maybe by other mechanisms) because once I freeze the plasmid, it won't work for IVT again.

Thank you again for your help! I hope my experience could help.