Quick Tips - Why don't my bands on a gel look correct after T7 digestion?

Often customers will ask "After a T7 digest, how come my bands don't run correctly on a gel?" There are two reasons why this could happen. One could be gel related and one could be due to the experiment.

If the bands look like they are running too high, it is usually due to T7 Endonuclease being a sticky enzyme. After digestion, you need to remove the protein from the DNA to prevent gel shifting. This can be done by incubating with Proteinase K to inactivate the T7 enzyme.

Another possibility is that the gene editing did not work and there are no digestion products visible. To confirm that the T7 enzyme is working, you would need a positive control. The EnGen mutation detection kit includes a control, so that you can be sure the T7 enzyme is working.

If you have any more questions, you can contact us at info@neb.com.