Jun 20, 2020
Version 1
Protocol for Exonuclease III (NEB #M0206) V.1
This protocol is a draft, published without a DOI.
- New England Biolabs1
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2020. Protocol for Exonuclease III (NEB #M0206). protocols.io https://protocols.io/view/protocol-for-exonuclease-iii-neb-m0206-7r9hm96
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28193
Keywords: exo III
Abstract
Exonuclease III efficiently degrades nicked and linear dsDNA (with blunt or 5' overhangs) from 3' to 5' direction, leaving supercoiled dsDNA intact.*
*Note: For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.
Materials
MATERIALS
EDTAThermo FisherCatalog #17892
Exonuclease III (E. coli)New England BiolabsCatalog #M0206
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Set-up the reaction as follows:
| COMPONENTS | 50 μl REACTION | |
| DNA | up to 5 μg | |
| NEBuffer 1 (10x) | 5 μl (1X) | |
| Exonuclease III | 0.5 μl (50 units) | |
| Nuclease-free H2O | up to 50 μl |
Incubate at 37 °C for 00:30:00 .
Stop reaction by adding EDTA to at least 11 millimolar (mM) .
Heat inactivate at 70 °C for 00:30:00 .
To clean up treated samples, we recommend using one of the following steps:
b. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), or
c. Performing a phenol/chloroform extraction followed by ethanol precipitation.