The following is a typical reaction protocol for nicking at the site of a single ribonucleotide within a dsDNA substrate or for nicking an Okazaki fragment 5´ to the ribonucleotide adjacent to the DNA
- Add 100 picomoles of double stranded substrate to 1X ThermoPol Buffer.
- Bring the reaction volume to 20 µl with water.
- Add 1 µl of RNaseHII and mix thoroughly.
- Incubate at 37°C for 15 minutes.
Note: To achieve the best results for a given substrate, it is often helpful to test more than one concentration of ribonuclease.