Getting Started
This protocol details how to assemble a standard PURE reaction to express protein from a suitable template DNA. This protocol is adapted and modified in many derivative protocols.
This guide uses pT7-plamGFP plasmid available in the Nucleus DNA Distribution (
DNA Distribution).
This protocol is currently built around the NEB PURExpress kit.
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Energy Mix (e.g., NEB PURE Solution A) | PURExpress® In Vitro Protein Synthesis Kit | New England Biolabs | E6800 | $295.00 | -85C to -75C | [link] |
Protein Mix (e.g., NEB PURE Solution B) | PURExpress® In Vitro Protein Synthesis Kit | New England Biolabs | E6800 | $295.00 | -85C to -75C | [link] |
RNAse inhibitor | RNase Inhibitor, Murine | New England Biolabs | M0314S | $81.00 | -25C to -15C | [link] |
Nuclease Free water | DEPC-Treated water | ThermoFisher Scientific | AM9916 | $147.00 | 4C to 30C | [link] |
PCR tube | Thin-walled, frosted lid, RNase-free PCR tubes | ThermoFisher Scientific | AM12225 | $205.00 | 4C to 30C | [link] |
DNA template (here: pT7-plamGFP) | User supplied | - | - | -25C to -15C | - |
Prerequisite Protocols
(optional) make your own Energy Mix: 
Make Energy Mix
Make Energy Mix(optional) make your own Protein Mix:
Assemble Protein Mix
Assemble Protein Mix
Protocol
Plan your reaction using the following Experiment Planning Template:
Component | Volume per Rxn (uL) | Total Volume (uL) |
Master Mix | (5x reactions) | |
NEB-Sol A | 4 | 20 |
NEB-Sol B | 3 | 15 |
RNAse Inb. | 0.5 | 2.5 |
pT7-plamGFP @100 fmol / uL | 0.5 | 2.5 |
Nuclease Free Water | 1 | 5 |
Total | 10 | 50 |
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Thaw the following reagents on ice:
Energy Mix (e.g., NEB PURExpress Solution A)
Protein Mix (e.g., NEB PURExpress Solution B)
RNAse Inhibitor
DNA template (e.g.,
pT7-plamGFP)Assemble PURE reactions by adding each component in the order they are listed in the Experiment Planning Template.
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Incubate samples at 37C ≥ 4 hrs.
(Optional): Read GFP fluorescence while incubating (see: 
Measuring Fluorescence with Plate Reader).
Measuring Fluorescence with Plate Reader).‣
Resources and References
Other Protocols
Papers
Original description of the PURE system - [Nature Biotechnology, 2001]
Resources
- Documentation from NEB for the PURExpress system [link]
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Developers
