Protocol for removing ssDNA from dsDNA or RNA Samples (M0568)

The protocol described below will enable degradation of up to 20 pmol of a 25 nt ssDNA (~200ng).  In order to degrade larger amounts of ssDNA or ssDNAs longer than 25 nt, we recommend adding more enzyme instead of extending the reaction time.  Users should note that ssDNAs longer than 25 nt may form secondary structures that hinder Thermolabile Exonuclease I activity.

1. Prepare a 20 ul reaction as follows:

Sample containing ssDNA (up to 20 pmol of a 25-mer) x μl
NEBuffer 3.1 (NEB #B7203)* 2 μl**
Thermolabile Exonuclease I  1 μl
Nuclease-free water              to 20 μl***

2. Incubate at 37°C for 4 minutes.

3. Stop reaction by heat-inactivation at 80°C for 1 minute.

*Most PCR buffers are compatible.  

**No reaction buffer is necessary if enzyme is added to a PCR reaction.

***Scale larger reaction volumes proportionally.