Assemble Base Cytosol

Overview¶

This protocol shows you how to assemble Base Cytosol from the following components: small molecule mix (SMix), tRNA, protein mix (PMix), ribosomes. To test the functionality of the assembled Cytosol we use the Reporter Module deGFP. Initial validation of the assembled Cytosol is documented in Yadav (2025).

Protocols and other resources are available for download at the bottom of this page.

Important Information

Please read this section carefully. It contains important notes, resources, and safety information. Not all information included here is included in the lab-ready protocol.

Notes

You can make Base Cytosol components yourself by following the Base Cytosol Processes
Base Cytosol components can be acquired as a premade reagent kit from b.next

Prerequisite Documentation

If you are preparing Base Cytosol components yourself, use the following protocols:
- Make Small Molecule Mix
- Make tRNAs
- Make Protein Mix
  - Protein Mix
  - OnePot Protein Mix
- Make Ribosomes

Genetically Encoded Components

Name	Type	Specification
`pOpen-deGFP`	Reporter	pOpen-deGFP

Composition

Component	Volume per Reaction ( $\mu$ L)
Small Molecule Mix (SMix)	3.0
tRNA	1.0
Protein Mix (PMix)	1.2
Ribosomes	1.8
RNase Inhibitor	0.5
`pOpen-deGFP`	0.5
User Additives	X
Nuclease Free Water	2.0 - X
Total	10

Materials and Equipment¶

This is an assembly process: it combines components produced by other Nucleus processes (Small Molecule Mix, Protein Mix, ribosomes, tRNAs, and the genetically encoded components listed above) rather than introducing new reagents. Its materials are itemized in those component processes’ Bills of Materials — see the consolidated Materials Reference.

Protocol¶

Cytosol Reaction Setup¶

Reaction Setup.

Component	Cytosol +deGFP DNA (µL)	Cytosol -deGFP DNA (µL)
SMix	10.5	10.5
Water	7	8.75
RNAse Inhibitor	1.75	1.75
PMix	4.2	4.2
tRNA	3.5	3.5
Ribosomes	6.3	6.3
`pOpen-deGFP` DNA template	1.75	0
Total master mix volume (µL)	35	35

Assemble Cytosol Reactions¶

Thaw reagents above on ice.

Chill two (2) PCR tubes on ice to assemble reaction master mixes.

Resuspend each component according to the following instructions:

Vortex SMix aggressively until visibly clear. Alternate 10s vortex / 10s rest on ice to maintain cool temperature. SMix should be transparent with no visible precipitate when ready.
Vortex or pipette mix tRNA.
Pipette mix PMix.
Do NOT vortex ribosomes! Gently pipette mix or flick the tube.

For each reaction, assemble reaction master mix in a chilled PCR tube by adding each reagent in the order and volume listed in the table above.

Mix the master mix thoroughly by pipette (6-10x) until visibly homogeneous.

Hold assembled reactions on ice or at 4°C until ready for measurement.

On a 384-well optical plate, array 10 µL of each reaction master mix into three (3) wells and note their locations.

In a plate reader set to 37°C, measure deGFP expression using the standard green fluorescence channel (ex: 485 nm, em: 515 nm).

Return reagents to their appropriate storage locations¶

Mark the lid of each Cytosol component that you used. The number of dots indicates how many freeze–thaw cycles each component has gone through.