Can we dilute PureExpress Cell Free Mix to increase number of reactions?
The PURExpress system from NEB is incredibly expensive, for this reason I have avoided it all costs during my experiments with cell-free systems.
This is the paper I've used to create my own cheaper cell-free systems while also discussing reasons why using the NEB PURExpress system is not recommended:
https://pubs.acs.org/doi/10.1021/acssynbio.8b00427

&quot;Although NEB provides a few different formulations of
the PURE system, the commercial system can’t be customized or optimized by the user, and the precise formulation of the commercial PURE system is not publicly available.&quot;

This might answer your question as to why I would not dilute the PURExpress system since the precise formulation is not publicly known. Thus, we can hypothesize what would work at various concentrations, but at the end of the day it is unknown what they specifically use in this reaction and how one could optimize it for their own purposes. It would take some trial and error if you really wanted to dilute/change the formulation.

&quot;Our method co-cultures and induces all 36 protein producing E. coli clones in a single flask followed by a single Ni-NTA purification.&quot;

This is the method I personally use and it can be time intensive, however the payoff is the cost and ability to optimize your system for your specific experiment.

&quot;The PURE system consists of several different components [7], that can be separated into three main categories: proteins (transcription, translation, and energy regeneration),ribosomes, and small molecule components (salts, buffers, NTPs, creatine phospate, and folinic acid)

If you were to go down the dilution route, I would suggest reaching out to NEB before hand to receive consultation for the purposes of your experiment and any consumables you may need to also purchase. This quote from the paper shows how your system has many considerations for consumables you may need to further research to maximize the expression of certain proteins. As for how this affects the reaction time, dilution shouldn't affect time, it will however affect the amount of product you get out of your reaction.