Prepare the transfer plate
Before you begin preparing your reagents, please ensure the Base Fluid has been connected to the instrument overnight. If the Base Fluid was not connected in advance, please open a new vial and incubate it for at least 1 hour at 30°C before use
Take all the reagents out of the freezer. The preparation of the transfer plate takes about 1 hour.
Our Quick Start User Guide can be used as a short guide tool how to prepare the transfer plate
The eProtein Discovery™ reagents need to be prepared and loaded onto a 96 well transfer plate following the layout and volumes in the figure and tables below.
It is critical to follow this layout exactly because it determines how the reagents are dispensed in the eProtein Discovery™ cartridge.
If an eGene™ construct is missing it must be substituted with 5 µL of Blank Buffer.
Do not substitute a missing eGene™ construct with water as this can have a significant negative impact on the droplet operations on the eProtein Discovery™ cartridge
eGene™ DNA construct
| Reagent | Well | Volume (µL) |
|---|---|---|
| eGene™ construct | A1 - A8 | 5 |
| eGene™ construct | B1 - B8 | 5 |
| eGene™ construct | C1 - C8 | 5 |
Reagents from NC3010
| Reagent | Well | Volume (µL) |
|---|---|---|
| Elution Buffer | H1-H2 | 10 |
| Blank Buffer | H3 - H4 | 10 |
| Universal Control | H5 | 10 |
| Complemantion Control | H6 | 10 |
| Workflow Control | H7 | 10 |
| Expression Control | H8 | 10 |
| Detector Protein | A10, B10, G10, H10 | 16 |
| Wash Buffer | C10, D10 | 16 |
| Elution Buffer | G10 | 16 |
| Cell-free Core (16 µL) + Additive 1 (2 µL) + Additive 2 (2 µL ) | 12A - 12H | 20 (16+2+2) |
Take the Strep Beads from the fridge and the Cartridge Kit reagents (box with the yellow stripe on the label) from the -80°C freezer.
Place an empty 96-well transfer plate on ice.
Apply the transfer plate sticker provided in the Cartridge Reagent kit and place the 96 well plate on ice.
The transfer plate should be kept on ice until the transfer of reagents to the Cartridge.
Ensure you prepare the Cell-free Blends last.
Be careful not to introduce any ice into the wells
eGene™ constructs (DNA)
Take the vials or the plates with the eGene™ constructs made in advance using the eGene™ Prep Kit, or provided by Nuclera through eGene™ Service, out of the freezer and thaw on the benchtop at room temperature. This takes approximately 15 minutes.
The vials or the plates should be centrifuged for a few seconds to ensure all the liquid is at the bottom of the wells.
It is critical not to leave any port empty. If a eGene™ construct is missing it must be substituted with 5 µL eGene Blank Buffer, not with water.
Tip: Empty ports can be used for duplicates.
Load 5 µL of each eGene™ construct onto the transfer plate into wells:
- A1 to A8
- B1 to B8
- C1 to C8
It is critical to load the eGene™ constructs onto the transfer plate in the exact order that they have been finalized in the experiment planned in the eProtein Discovery™ Cloud Software if using a Cloud Connected System.
eProtein Discovery™ purification reagents
Thaw the Wash Buffer and the Elution Buffer on the benchtop at room temperature. Once thawed, vortex for 2 seconds and centrifuge for 2 seconds using a microcentrifuge to mix and collect all the reagents.
- Load 16 µL of Wash Buffer into wells C10 and D10.
- Load 16 µL of Elution Buffer into well F10
- Load 10 µL of ELution Buffer into wells H1 and H2
eProtein Discovery™ controls
From the kit kept at -80°C, take the controls out and thaw them on ice.
- Load 10 µL of Blank Buffer into wells H3 and H4.
- Load 10 µL of Universal control into well H5.
- Load 10 µL of Complementation Control into well H6.
- Load 10 µL of Full W/F Control into well H7.
- Load 10 µL of Expression Control into well H8
Strep Purification Beads
Beads settle extremely quickly. In all following steps, do not pause between resuspending and taking aliquots. To obtain a proper aliquot, gently pipette up and down 10 times with and then immediately pipette up an 11th time to take the aliquot.
Strep Purification Beads are provided in 200 µL aliquots of 5% v/v suspension – To prepare the Strep Beads:
- Take the vial of Strep Beads from the fridge and give it a quick spin for 2 seconds in a microcentrifuge to pellet the beads.
- Resuspend the beads by gently pipetting up and down 10 times with a p200 pipette set on 90 µL.
- Transfer 90µL of the resuspended beads into a 1.5 mL tube. Discard the rest only after the experiment starts, in case more volume is required.
- Place the tube with Strep Beads on a magnetic particle separator and capture for 30 sec.
- Remove all the supernatant with a p200 pipette and discard the liquid.
- Remove the tube with Strep Beads from the magnetic particle separator. Resuspend the beads in 100 µL Wash Buffer by slowly pipetting up and down 10 times.
- Repeat steps 4 to 6 twice more for a total of three washes.
- After the third wash, spin down the tube and place it back on a magnetic particle separator and capture for 30 sec
- Remove all the supernatant with a p200 pipette and discard the liquid.
- Spin down the tube, place it back on a magnetic particle separator and remove the residual buffer with a p20 pipette.
- With a p20 pipette, resuspend the beads in 10.5 µL Wash Buffer by gently pipetting up and down 10 times to create a 15 µL 30% Strep Beads working
- Keep the beads in the tube on the bench, not on ice.
The beads should NOT be loaded onto the transfer plate.
Detector Protein
The Detector Protein is supplied as ready to use. Spin down the tube for 2 seconds to collect the full volume at the bottom. Load 16 µL of Detector Protein into wells A10, B10, G10, and H10 of the transfer plate.
Preparation of the Cell-free Blends
For each expression screening experiment, up to eight 20 μL Cell-free Blends can be made by adding 16 μL of Cell-free Core Reagent, 2 μL of a first additive, and 2 μL of a second additive.
The total volume of blend should always be 20 µL final
The same additive can be used as first and second additive, for example 2 x 2 µL of Additive Buffer.
Ensure that the Cell-free blend is thoroughly resuspended by pipetting up and down from near the bottom.
- Thaw on ice Cell-free Core Reagents and Additives
- Once thawed, vortex for 2 seconds the Cell-free Core reagents and Additives to ensure they are well mixed.
- Centrifuge for 2 seconds the Cell-free Core reagents and Additives using a microcentrifuge to return any droplets to the bulk aliquot.
- Add 16 µL of Cell-free Core reagent to wells A12-H12.
- Add 2 µL of your first selected additive to wells A12-H12.
- Add 2 µL of your second selected additive to wells A12-H12.
It is critical to load the Cell-free Blends onto the transfer plate in the exact order that they have been finalized in the experiment planned in the eProtein Discovery software.
Ensure the Cell-free Blend is thoroughly resuspended with the chosen additives by pipetting up and down near the bottom of the tube, making sure that any viscous components are fully mixed.