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


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


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.

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