Nicking a single ribonucleotide site within a dsDNA substrate or an Okazaki fragment with RNase HII (NEB #M0288)

Overview

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

Protocol

Reaction mix:

1. Resuspend 100 pmol of double-stranded substrate into 50 μl of 1X ThermoPol Buffer.
2. Add 1 μl of RNase II and mix thoroughly.
3. Incubate at 37°C for 30 minutes. 
   
   Note: To achieve the best results for a given substrate, it is often helpful to test more than one concentration of ribonuclease.