Assemble PURE Reactions

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 the pT7-GFP plasmid available in the Nucleus DNA Distribution (DNA Distribution). The T7 promoter makes it optimized for expression in the PURE system.

This protocol is currently built around the PURExpress kit obtained from New England Biolabs.

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Link
PURE	PURExpress® In Vitro Protein Synthesis Kit	New England Biolabs	E6800	$295.00	[link]
RNAse inhibitor	RNase Inhibitor, Murine	New England Biolabs	M0314S	$81.00	[link]
Nuclease free water	DEPC-Treated water	ThermoFisher Scientific	AM9916	$147.00	[link]
PCR tube	Thin-walled, frosted lid, RNase-free PCR tubes	ThermoFisher Scientific	AM12225	$205.00	[link]
pT7-GFP (or other suitable DNA template)	User supplied	DNA Distribution	-	-	-

Prerequisite Protocols

This protocol can be completed without reference to other protocols if using the PURExpress kit obtained from New England Biolabs (NEB). However, we have validated an energy solution that can be substituted for NEB PURExpress Solution A. A substitute for NEB PURExpress Solution B is forthcoming.

Set the concentration of your template DNA to 10 fmol/uL.

(optional) prepare 2.5x Energy Mix:

Make Energy Mix .

Protocol

Plan your reaction using the following Experiment Planning Template:

Component	Reaction Volume (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-GFP	0.5	2.5
Total	9	45

Note: in general, PURE reactions should be 10 uL but can be run with an additional 20% volume (i.e. 12 uL). Descending the table describes the order in which components should be added when constructing your reaction.

Note: pT7 -GFP is included only as an example. Any other with a T7 promoter will be compatible. We standardize the concentration of template stock solution to 10 fmol/uL and use between 0.5 and 1.0 uL per reaction.

Thaw the following reagents on ice:

NEB PURExpress Solution A (stored at - 80C)

NEB PURExpress Solution B (stored at - 80C)

RNAse Inhibitor (stored at - 20C)

pT7-GFP DNA (stored at -20C)

Optional: any additional components referenced by hyperlinked protocols.

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Note

Assemble PURE reactions in the order they are listed in the Experiment Planning Template.

Each reagent tube containing a protein or template DNA should be agitated before assembling the reactions to dislodge proteins from container walls and to remix

Make sure to pipette mix the final assembled reaction thoroughly since the resulting mixture is highly viscous

Incubate samples at 37C for at least ≥ 4 hrs.

(Optional): Read GFP fluorescence while incubating (see:

Measuring Fluorescence with Plate Reader).

Clean up

We find it useful to keep track of the number of freeze-thaw cycles PURExpress solution A and B have been subjected to. We simply add a dot to a conspicuous location on the tube each time the material has been thawed. In our experience, results are consistent for up to 5 freeze-thaw cycles. If your experiments are such that the tube will last for more than 5 freeze-thaw cycles, aliquot your samples to an appropriate volume to minimize the number of cycles they are subjected to.