Jun 20, 2020
Version 1
Protocol for T7 Exonuclease (NEB #M0263) V.1
This protocol is a draft, published without a DOI.
- 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 T7 Exonuclease (NEB #M0263). protocols.io https://protocols.io/view/protocol-for-t7-exonuclease-neb-m0263-7r7hm9n
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: June 20, 2020
Protocol Integer ID: 28191
Abstract
T7 Exonuclease efficiently degrades nicked and linear dsDNA (with blunt or 3' overhangs) from 5' to 3' direction, leaving supercoiled dsDNA inctact.*
*Note: For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.
Materials
MATERIALS
EDTAThermo FisherCatalog #17892
T7 ExonucleaseNew England BiolabsCatalog #M0263
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 1 μg | |
| NEBuffer 4 (10x) | 5 μl (1X) | |
| T7 Exonuclease | 1 μl (10 units) | |
| Nuclease-free H2O | up to 50 μl |
Incubate at 25 °C for 00:30:00 .
Stop reaction by adding EDTA to at least 11 millimolar (mM) .
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.