Every in vitro transcription (IVT) reaction starts with DNA you put there on purpose. A linearized plasmid or polymerase chain reaction (PCR) template goes in, the polymerase does its work and a few hours later you have RNA, often a lot of it. The template’s job is done.

In theory, a quick DNase step takes care of the rest.

In practice, the template doesn’t always leave quietly.

The DNA you added is the hardest DNA to remove

In an IVT reaction, the DNA was added deliberately, often in microgram amounts. It sits in a buffer designed to make the polymerase as productive as possible, which is not the same as making DNase productive.

That’s the trade-off at the center of IVT cleanup. The easiest place to digest the template is the reaction tube, right after transcription, with no extra purification step. But IVT reactions carry high ionic strength and a crowded mix of other components, conditions that suppress the activity of many DNase I enzymes (1,2). The reaction is good at making RNA and bad at getting rid of what made it.

Continue reading “The Template That Stayed Behind: Why DNA Removal After In Vitro Transcription Is Harder Than It Looks”