Digestion for BioBrick Assembly Kit (E0546)
New England Biolabs
Published: 2022-02-03 DOI: 10.17504/protocols.io.bddsi26e
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Abstract
This protocol explains methods for assembling multi-compontent genetic systems using BioBrick® parts.
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.
Steps
1.
Digest Upstream Part with EcoRI-HF® and SpeI:
| A | B |
|---|---|
| Reagent | Volume |
| Upstream Part Plasmid | 500 ng |
| EcoRI-HF | 1 µl |
| SpeI | 1 µl |
| 10X NEBuffer 2.1 | 5 µl |
| H2O | to 50 µl |
2.
Digest Downstream Part with XbaI and PstI:
| A | B |
|---|---|
| Reagent | Volume |
| Upstream Part Plasmid | 500 ng |
| XbaI | 1 µl |
| PstI | 1 µl |
| 10X NEBuffer 2.1 | 5 µl |
| H2O | to 50 µl |
3.
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.
Digest the Destination Plasmid with EcoRI-HF® and PstI:
| A | B |
|---|---|
| Reagent | Volume |
| Destination Plasmid DNA | 500 ng |
| EcoRI-HF | 1 µl |
| PstI | 1 µl |
| 10X NEBuffer 2.1 | 5 µl |
| H2O | to 50 µl |
Note
4.
Incubate all three restriction digest reactions at 37°C for 0h 10m 0s.
5.
Heat inactivate at 80°C for 0h 20m 0s.
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