Assemble PURE Reactions

<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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This protocol details how to assemble a standard 10 uL 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.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Energy Mix	Solution A: PURExpress® In Vitro Protein Synthesis Kit	New England Biolabs	E6800	$295.00	-85C to -75C	[link]
Protein Mix	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

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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[ ] (optional) make your own Energy Mix: Make Energy Mix
[ ] (optional) make your own Protein Mix: Make Protein Mix

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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[ ] Plan your reaction using the following Experiment Planning Template:

Component	Volume per Rxn (uL)	Total Volume (uL)
Master Mix		e.g., for 5x reactions
Energy Mix (e.g., NEB Sol A)	4	20
Protein Mix (e.g., 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

Note: you can run PURE reactions up to 20% over volume
Note: your DNA template should use pT7 as a promoter

[ ] 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 listed in the Experiment Planning Template.
- Note: mix your components and final reaction
[ ] Incubate samples at 37C ≥ 4 hrs.
[ ] (Optional): Read GFP fluorescence while incubating (see: Measuring Fluorescence with Plate Reader).
- Note: keep track of freeze-thaw cycles

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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<aside> <img src="/icons/document_gray.svg" alt="/icons/document_gray.svg" width="40px" /> Other Protocols

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<aside> <img src="/icons/book-closed_gray.svg" alt="/icons/book-closed_gray.svg" width="40px" /> Papers

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Original description of the PURE system - [Nature Biotechnology, 2001]

<aside> <img src="/icons/info-alternate_gray.svg" alt="/icons/info-alternate_gray.svg" width="40px" /> Resources

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Documentation from NEB for the PURExpress system [link]

<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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