Feb 02, 2022

Public workspaceDigestion for BioBrick Assembly Kit (E0546) V.2

DOI
dx.doi.org/10.17504/protocols.io.bddsi26e
  • 1New England Biolabs
  • New England Biolabs (NEB)
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DOI: dx.doi.org/10.17504/protocols.io.bddsi26e
External link: https://www.neb.com/protocols/0001/01/01/digestion-protocol-e0546
Protocol Citation: New England Biolabs 2022. Digestion for BioBrick Assembly Kit (E0546). protocols.io https://dx.doi.org/10.17504/protocols.io.bddsi26eVersion created by New England Biolabs
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: March 08, 2020
Last Modified: February 02, 2022
Protocol Integer ID: 33938
Keywords: Biobrick, Assembly, E0546,
Abstract
This protocol explains methods for assembling multi-compontent genetic systems using BioBrick® parts.
Guidelines
The BioBrick® Assembly Kit provides a streamlined method for assembly of BioBrick parts into multi-component genetic systems. BioBrick parts are DNA sequences that encode a defined biological function and can be readily assembled with any other BioBrick part. The process for assembling any two BioBrick parts is identical and results in a new composite BioBrick part.

The BioBrick Assembly Kit contains EcoRI-HF®, XbaI, SpeI, PstI, T4 DNA Ligase and NEBuffer 2.1.

BioBrick Assembly Overview
Materials
MATERIALS
ReagentBioBrick Assembly Kit - 50 rxnsNew England BiolabsCatalog #E0546S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
The BioBrick® Assembly Kit was developed in partnership with Ginkgo BioWorks. What follows is an abbreviated set of protocols for the use of the BioBrick® Assembly Kit (to assemble an Upstream Part with a Downstream Part into a Destination Plasmid). For more details and for technical questions, please see here.
Digest Upstream Part with EcoRI-HF® and SpeI:
AB
ReagentVolume
Upstream Part Plasmid500 ng
EcoRI-HF1 µl
SpeI1 µl
10X NEBuffer 2.15 µl
H2Oto 50 µl

Digestion
Digest Downstream Part with XbaI and PstI:
AB
ReagentVolume
Upstream Part Plasmid500 ng
XbaI1 µl
PstI1 µl
10X NEBuffer 2.15 µl
H2Oto 50 µl

Digestion
Digest the Destination Plasmid with EcoRI-HF® and PstI:
AB
ReagentVolume
Destination Plasmid DNA500 ng
EcoRI-HF1 µl
PstI1 µl
10X NEBuffer 2.15 µl
H2Oto 50 µl

Note
The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdB, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

Digestion
Incubate all three restriction digest reactions at Temperature37 °C for Duration00:10:00 .
Incubation
Heat inactivate at Temperature80 °C for Duration00:20:00 .
Incubation