Feb 18, 2022
Version 2
Blunting Protocol for NEB PCR Cloning Kit (E1202) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2022. Blunting Protocol for NEB PCR Cloning Kit (E1202). protocols.io https://dx.doi.org/10.17504/protocols.io.bfhnjj5eVersion created by New England Biolabs Tech Support
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: April 23, 2020
Last Modified: February 18, 2022
Protocol Integer ID: 36110
Keywords: blunting, cloning, NEB PCR cloning kit, E1202,
Abstract
This is the blunting protocol for NEB PCR Cloning Kit (E1202).
Guidelines
Reaction volume may be scaled up or down as necessary.
PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1.1, 2.1, 3.1, and CutSmart™ Buffer in addition to NEBuffers 1-4, BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 and 1.1 where the total yield is about 50% of optimum.
Materials
MATERIALS
NEB PCR Cloning Kit - 20 rxnsNew England BiolabsCatalog #E1202S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Reaction volume may be scaled up or down as necessary.
Mix the following components in a sterile microfuge tube:
| A | B | |
| COMPONENT | AMOUNT | |
| Purified DNA (up to 5 µg) | 1-19 µl | |
| 10X Blunting Buffer | 2.5 µl | |
| 1 mM dNTP Mix | 2.5 µl | |
| Blunt Enzyme Mix | 1.0 µl | |
| Sterile dH20 | variable | |
| Total volume | 25 µl |
Determine whether your reactions are using DNA digested by restriction enzymes or are sheared/nebulized or PCR products and move forward with the following steps:
Note
PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
Note
Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1.1, 2.1, 3.1, and CutSmart™ Buffer in addition to NEBuffers 1-4, BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 and 1.1 where the total yield is about 50% of optimum.
Restriction enzyme DNA3 steps
Incubate the reactions containing restriction enzyme digested DNA at Room temperature for 00:15:00 .
Immediately inactivate enzyme in the blunting reaction by heating at 70 °C for 00:10:00 .
Proceed directly to the ligation step using the Quick Ligation Kit (NEB #M2200) or standard T4 DNA Ligase (NEB #M0202).
Note
Blunt ligation reactions using standard T4 DNA Ligase should be incubated Overnight at Room temperature .