Assay development guidelines and protocol notes
This topic explains the following:
• Each step of the protocol so that you can optimize the On‑Cartridge Reaction protocol to your particular experimental design
• Automation movements during the protocol
For details on how to use the Experiments Editor, see Using Experiment IDs.
Protocol stepwise guidelines
Protocol step | Guidelines and notes |
|---|---|
Number of Full Columns of Cartridges | This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: • Greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. • Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup. Default: 1 Range: 1-12 |
Initial Syringe Wash | This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Equilibrate | This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for the reaction during the Reaction step. In the case of an enzymatic reaction, the equilibration buffer would be the optimized enzyme reaction buffer. Ensure that the optimal reaction buffer does not break the affinity interaction holding the immobilized substrate on the resin bed. In preparation for equilibration, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the wash station, 10 µL of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (µL). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. • Volume for 5 µL cartridge: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate. A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min using the default volume may not equilibrate through the pores in the beads. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10 |
Collect Flow Through | This step is used for optimizing or troubleshooting a protocol to ensure that the equilibration solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the dispenses the flow-through during the Equilibrate step into the Flow Through Collection plate for downstream processing or for analysis. If this step is not selected, the flow-through from the Equilibrate step is dispensed directly into the wash station. This step is not selected by default. |
Reaction | This step enables the reaction in the cartridge resin bed. No liquid is removed or added to the cartridge cups before the reaction begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Reaction step aspirates the Reagent through the cartridges in two steps, and then aspirates a chase volume: 1 Initial Aspiration. Aspirates the Initial Draw volume at a flow rate of 10 µL/min. 2 Secondary Aspiration. Aspirates any additional volume at a flow rate appropriate to satisfy the Duration setting. For example, if the volume is set at 6 µL and the time was set for 30 minutes, the remaining 2 µL of the 6 µL volume would be aspirated at a flow rate of ~0.068 µL/min (2 µL / (30 min – 0.4 min)). 3 Reaction Chase Aspiration. Aspirates the chase at the specified volume and flow rate. The Reaction flow-through and Reaction Chase are collected in the syringes, and then dispensed into the Flow Through Collection plate, unless Combine with Eluate is selected. In which case, the Reaction flow-through and the Reaction Chase are dispensed into the Elution plate. This step is selected by default. Volume (µL). The volume must be determined empirically. For an enzymatic reaction, the volume would depend on the enzyme. If the enzyme is robust enough to undergo the number of enzymatic cycles required to push the reaction to completion, the Initial Draw volume may be sufficient. If the enzyme undergoes a limited number of cycles, the enzyme in the resin bed may need to be replenished during the course of the reaction, and significantly more than the Initial Draw volume may be required. • Volume for 5 µL cartridge: – Default: 6 – Practical: 4–15 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 30 – Practical: 20–75 – Range: 0–250 Flow Rate. The initial draw volume is aspirated at 10 µL/min. Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting. Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Scenario using default settings for 5 µL cartridges: 1 The Peltier Thermal Station heats until the set point temperature ±2°C is reached. 2 The Bravo 96AM Head picks up the cartridges from the Cartridge & Tip Seating Station. 3 The Reagent is aspirated through the cartridges: 4 µL at a flow rate of 10 µL/min and 2 µL at a flow rate of 0.068 µL/min. (The Duration of both aspiration steps is 30 minutes.) 4 An external cartridge wash is conducted at the wash station. 5 15 µL Chase Buffer is aspirated at 5 µL/min. 6 The cartridges are ejected into the Cartridge & Tip Seating Station. 7 The syringe contents are dispensed into the Flow Through Collection plate. 8 Three internal syringe wash cycle are conducted. | |
Temperature | This setting specifies the set point temperature of the Peltier Thermal Station during the Reaction step. The optimal temperature is a function of the reaction being conducted. The actual temperature in the cartridge will be less than this setting. Heat loss occurs as the heat transfers across the thermal plate insert, a small air gap between the plate insert and the wells of the plate, the plastic of the plate, the solution in the well, and the cartridge resin bed. For example, a temperature setting of 45 °C results in a cartridge bed resin temperature of approximately 37 °C using the Red PCR Plate Insert and a PCR plate. A thermal plate insert is critical when running a reaction at a temperature other than room temperature. Without the insert, the air gap between the Thermal Station and the wells of the plate results in a greater temperature differential between the setting in the application and the actual temperature in the cartridge bed. The temperature differential is not a constant percentage of the set point, so the preceding example should be considered only as a starting point for cartridge target temperatures other than 37 °C in the PCR plates. In addition, plate types vary in heat transfer efficiency because of their differences in design, such as well shape.  At least 3–5 µL of liquid is required in each well of a 96-well PCR plate at the end of the incubation time as this is the minimum volume required to maintain contact with the bottom of the cartridges. This contact is required to conduct heat into the resin bed. Longer incubation times or higher temperature settings require more volume at the beginning of the run to account for added evaporation. This volume should be determined empirically. A PCR plate is the most practical and common plate type at deck location 4 because of the low volumes of solution typically used at that location. The minimum volume required in the wells at the end of the incubation time for other plate types must be determined empirically. Example using Red PCR Plate Insert plus PRC plate. To conduct a 30-minute reaction at a resin bed temperature of approximately 37 °C with a 6 µL total aspiration volume, the temperature should be set to 45 °C and 12 µL of enzyme solution should be added per well before the run is started. 12 µL is required because 6 µL will be aspirated, 3 µL are expected to evaporate, and 3 µL are required at the end of the run to ensure heat conductance occurs during the entire run. Temperature °C: • Default: 25 • Practical: 20–60 • Range: 4–110 |
Duration | This setting specifies the total length of time to aspirate the Reagent through the cartridges, including the initial and secondary aspiration steps. Note: This setting governs only the aspiration of Reagent through the cartridges. It does not include the time required to ramp up to temperature or the time required for the Reaction Chase step. Note: The duration of the reaction is related to the concentration of the reactant. In general, as the concentration of the reactant (for example, enzyme) decreases, the duration increases, as does the amount of reaction solution lost during the reaction due to evaporation, and the volume of excess reaction solution required. See the notes in the preceding Temperature step for more details. Time (minutes): • Default: 30 • Practical: 5–60 • Range: 0–180 |
Initial Draw | Specifies the initial draw volume, which is aspirated at 10 µL/min. Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting. Volume (µL): • Volume for 5 µL cartridge: – Default: 4 – Practical: 3–9 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 20 – Practical: 15–45 – Range: 0–250 Note: Setting the volume to zero skips all Reaction tasks except syringe washing. |
Reaction Chase | This step washes the soluble reaction products out of the resin bed and into the syringes using the Chase Buffer. The Reaction Chase occurs immediately after the aspiration of Reagent, combining the flow-through of the initial and secondary aspiration and the Chase Buffer within the syringes. Volume (µL). Elution of products off the AssayMAP cartridges is typically 2–3 column volumes when the affinity between the eluate and resin has been completely disrupted. Therefore, 10–15 µL (5 µL cartridge) or 50–75 µL (25 µL cartridge) should be a sufficient chase volume as long as there is no significant affinity between the soluble reaction product and the resin bed. The default volume is set to 3 column volumes to be conservative, but it is likely that you can decrease this volume. • Volume for 5 µL cartridge: – Default: 15 – Practical range: 15–30 – Range: 0–250 • Volume for 25 µL cartridge: – Default: 75 – Practical range: 50–100 – Range: 0–250 Note: Setting the volume to zero skips all Reaction tasks except syringe washing. Flow Rate: • Default: 5 • Practical: 5–20 • Range: 0.1–500 |
Combine with Eluate | This setting specifies where the combination of the Reaction initial and secondary aspiration and the Chase Buffer is collected: • If selected, collects the Reaction initial and secondary aspiration and chase in the Eluate Collection plate. This is a good choice if the downstream analysis can easily separate the reactant, soluble, and eluted reaction products, allowing all the reaction products to be analyzed in a single run. • If not selected, collects the Reaction initial and secondary aspiration and chase in the Flow Through Collection plate. This is a good choice if the goal is to analyze the soluble and immobilized reaction products separately because – Data on only one of these components is required, and there is not a good or rapid way of chromatographically separating the reactant, buffer, soluble, and immobilized reaction products in a single analytical run. – One wants to remove the buffer, reactant, and soluble reaction product from the immobilized reaction product, and elute this immobilized reaction product in a mass-spec-compatible solution. The setting is not selected by default. |
Cup Wash 1 | This step removes the residual liquid that may remain above the resin bed after the Reaction step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (µL). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 0–100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 1 | This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. In preparation for Internal Cartridge Wash 1, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the wash station, 10 µL of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–100 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate. A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may not equilibrate through the pores in the beads, resulting in incomplete washing. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | This step is typically used for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the flow-through from Internal Cartridge Wash 1 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed into the wash station. This step is not selected by default. |
Cup Wash 2 | This step removes the residual buffer that may remain above the resin bed after the Internal Cartridge Wash 1 step. This step aspirates Cartridge Wash Buffer 2 and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station. The syringes are washed at the wash station. This step is not selected by default. Volume (µL). A volume less than the default may be insufficient for cup washing, while a volume >50 µL may offer little benefit. • Default: 25 • Practical: 25–50 • Range: 0–100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. • Default: 3 • Practical: 3–5 • Range: 0–10 |
Internal Cartridge Wash 2 | This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Cartridge Wash Buffer 1 from the resin bed. In preparation for Internal Cartridge Wash 2, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid that is in the cups is removed by a 60 µL aspiration, the aspirated solution is discarded at the wash station, 10 µL of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer from the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is not selected by default. Volume (µL). Volumes higher than the default volume (10 column volumes) has little benefit but will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. • Volume for 5 µL cartridges: – Default: 50 – Practical: 50–150 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 250 – Practical: 250 – Range: 0–250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate. A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may not equilibrate through the pores in the beads, resulting in incomplete washing. • Default: 10 • Practical: 5–20 • Range: 0.5–500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 µL of deionized water is used for each syringe wash cycle. • Default: 3 • Practical: 2–5 • Range: 0–10 |
Collect Flow Through | This step is typically only used for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the liquid eluted during Internal Cartridge Wash 2 is dispensed directly into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed directly into the wash station. This step is not selected by default. |
Stringent Syringe Wash | This step cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (µL). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. • Default: 50 • Practical: 50–100 • Range: 0–250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. • Default: 2 • Practical: 2–5 • Range: 0–10 |
Elute | This step uses Elution Buffer to elute immobilized reaction products from the cartridges. In preparation for elution, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the wash station, 10 µL of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the eluate is mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 µL, the samples will not be mixed. You can also select the Eluate Discard and Existing Collection Volume substeps, which are described in the following rows of this table. This step is selected by default. Volume (µL). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 2–3 column volumes (10–15 µL for 5 µL cartridges, or 50–75 µL for 25 µL cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume), the Reaction and Reaction chase volumes if Combine With Eluate Volume is selected, and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. • Volume for 5 µL cartridges: – Default: 25 – Practical: 10–30 – Range: 0–250 • Volume for 25 µL cartridges: – Default: 125 – Practical: 50–150 – Range: 0–250 Note: Setting the volume to zero skips all Elute tasks except syringe washing. |
Flow rate. A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 µL/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). • Default: 5 • Practical: 2–15 • Range: 0.1–500 Wash cycle. The number of syringe washes to perform at the wash station after an Elute step. 250 µL of DI water is used for each syringe wash cycle. • Default: 1 • Practical: 1–3 • Range: 0–10 | |
Eluate Discard | This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. The Eluate Discard volume is dispensed into the wash station. The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate into the Eluate Collection plate. Example: If the Elute and Eluate Discard steps are selected with the following settings, Elute volume = 15 µL (5 µL cartridges) or 50 µL (25 µL cartridges) Eluate Discard volume = 2 µL (5 µL cartridges) or 10 µL (25 µL cartridges) the first 2 µL (5 µL cartridges) or 10 µL (25 µL cartridges) eluate from the cartridges will be discarded to the wash station, and the remaining 13 µL (5 µL cartridges) or 40 µL (25 µL cartridges) eluate will be collected in the Eluate Collection plate. Select the Eluate Discard step in a situation where minimizing the volume of eluate is critical. For AssayMAP cartridges, the initial elution volume (~2 µL for 5 µL cartridges and ~10 µL for 25 µL cartridges) contains small or no measurable amounts of analyte. This step is not selected by default. Volume (µL). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. • Default: 0 • Practical: – 5 µL cartridges: 0–2 – 25 µL cartridges: 0–10 • Range: 0–250 |
Existing Collection Volume | This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume, the net volume from the Elute step (Elute volume - Eluate Discard volume), and the soluble reaction products, if Combine With Eluate is selected, feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume-dependent manner. For the maximum practical working volumes of labware for eluate collection, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (µL): • Default: 0 • Practical: 0–250 • Range: 0—1000 Note: Total elution collection well volumes >500 µL may require additional off-deck mixing to reach homogeneity. |
Final Syringe Wash | This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 µL of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 µL aspiration and then discarded into the wash station. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 µL of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: • Default: 3 • Practical: 3–5 • Range: 0–10 |
Automation movements during the protocol
This section describes the basic movements of the AssayMAP Bravo Platform during the On-Cartridge Reaction protocol using the default method. Changing the selections or parameters will alter the movements.
Protocol step | Head moves to deck location... | Action |
|---|---|---|
Start protocol | – | Sets Peltier Thermal Station to set point temperature (°C). |
2 | Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted. | |
1 | Dispenses any liquid remaining in the syringes into the wash station. | |
Initial Syringe Wash | 1 | Washes the syringes the specified number of cycles. |
Equilibrate | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
1 | Dispenses into the wash station between the chimneys, and then performs an external probe wash. | |
3 | Aspirates 10 µL of Equilibration Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of Equilibration Buffer into the cartridge cups and exercises the cartridges off task. | |
3 | Aspirates the Equilibration Buffer. | |
2 | Mounts the cartridges on the head. | |
1 | Dispenses the Equilibration Buffer through the cartridges to equilibrate. Washes the cartridge exteriors. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Reaction | 1 | Waits for Peltier Thermal Station to reach ±2 °C of the Reaction Temperature setting. |
2 | Mounts the cartridges on the head. | |
4 | Aspirates Initial Draw volume at 10 µL/min. | |
4 | Aspirates the remaining volume (Reaction Volume setting in the form minus Initial Draw volume) for the specified Duration (minutes) at the set Temperature (°C). | |
1 | Washes the cartridge exteriors at the wash station. | |
3 | Aspirates the specified volume of Chase Buffer at the specified Flow Rate (µL/min). | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
7 | Dispenses the flow-through into the Flow Through Collection plate. Mixes the flow-through. | |
1 | Washes the syringes. | |
Cup Wash 1 | 5 | Aspirates cartridge Cartridge Wash Buffer 1 into the syringes. |
2 | Washes the cartridge cups and exercises the cartridges off task. | |
1 | Dispenses the buffer into the wash station between the chimneys. | |
1 | Washes the syringes. | |
Internal Cartridge Wash 1 | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
1 | Dispenses into the wash station between the chimneys, and then performs an external probe wash. | |
5 | Aspirates 10 µL of Cartridge Wash Buffer 1 for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
5 | Aspirates Cartridge Wash Buffer 1 into the syringes for the Internal Cartridge Wash 1 step. | |
2 | Mounts the cartridges on the head. | |
1 | Dispenses Cartridge Wash Buffer 1 through the cartridges at the Internal Cartridge Wash 1 flow rate. | |
1 | Washes the exterior of the cartridge tips. | |
2 | Parks the cartridges in the seating station. | |
1 | Washes the syringes. | |
Stringent Syringe Wash | 8 | Aspirates the Syringe Wash Buffer (Elution Buffer). |
1 | Dispenses the buffer at the wash station. | |
1 | Washes the syringes. | |
Elute | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
1 | Dispenses into the wash station between the chimneys, and then performs an external probe wash. | |
8 | Aspirates 10 µL of Elution Buffer for the cartridge air-gap-prevention step. | |
2 | Dispenses the 10 µL of buffer into the cartridge cups and exercises the cartridges off task. | |
8 | Aspirates the Elution Buffer. | |
2 | Mounts the cartridges. | |
9 | Elutes the samples into the Eluate Collection plate. | |
1 | Washes the cartridge exteriors. | |
2 | Parks the cartridges in the seating station. | |
9 | Mixes eluates. | |
1 | Washes the syringes. | |
Final Syringe Wash | 2 | Aspirates 20 µL of air above this location, moves down to just above the cartridge engagement point and aspirates 60 µL, and then exercises the cartridges off task. |
1 | Dispenses into the wash station between the chimneys. | |
1 | Washes the syringes. |