Assay development guidelines
This topic provides guidelines for adjusting the protocol, and notes about the application.
Note: For maximum well capacities and overage recommendations in the serial dilution plates, see the Labware Reference Guide in the Literature Library of the Protein Sample Prep Workbench.
Automation movements and stepwise guidelines
This section describes the basic automation movements of the AssayMAP Bravo Platform during the Serial Dilution run.
Protocol step | Head moves to deck location… | Action |
|---|---|---|
1. Syringe Wash | 1 | Performs 1 external syringe wash at the wash station. |
2. Syringe Drying | 1 | Performs 4 syringe aspirate-and-dispense cycles above the wash station to cycle air in and out of the syringes. The syringes move over the chimneys after each cycle to remove any droplets that were pushed out of the syringes during the cycle. |
3. Initial Tip Transfer | 3 | Presses on all the 250-µL pipette tips from the tip box. Note: The tip box must be full even when using fewer than 96 pipette tips because the tip-seating force has been set assuming that a full rack of pipette tips are being seated. |
5 | Ejects the pipette tips into the seating station. | |
4. Offset Head Tip Pickup | 5 | Positions the head at an offset from the seating station to pick up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips. • Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. • Picks up tips in column format for left-to-right or right-to-left dilution. |
5. Diluent Transfer | 2 | Moves to the diluent reservoir. • If a blowout volume is specified, aspirates an air gap. This volume is used to ensure that no liquid remains in the pipette tip after the contents of the tip are dispensed. A small blowout volume can help improve precision and accuracy. The magnitude of this effect is usually small for aqueous solutions but can be significant with viscous solutions. Default: 5 µL Range: 0–50 µL Note: The size of the air gap reduces the possible pipetting volume per pipetting cycle. Larger air gaps have diminishing returns in terms of improved accuracy and precision. The relationship between the size of the air gap and the precision and accuracy is highly dependent on the solution being moved. Agilent recommends starting with the default value to determine if any change is required. • If specified, prewets the pipette tips in the designated source wells. Prewetting pipette tips is best practice for good pipetting accuracy and precision. Default: Yes Options: Yes, No • Aspirates diluent into the pipette tips. |
6 | • Dispenses the diluent into the designated wells in the serial dilution plate. • If a blowout volume is specified, the head raises to near the top of the well, and the specified volume of air is dispensed followed by a tip touch on the east and west sides of the well. This aspirate-and-dispense process repeats using the same pipette tips until all the wells in the serial dilution plate have the specified amount of diluent. If a blowout is specified, an air gap is aspirated at the start of each aspirate task and a blowout and tip touch occur after each dispense task. | |
6. Change Tips | 3 | Ejects the used pipette tips into the tip box. |
5 | Picks up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips in the seating station. • Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. • Picks up tips in column format for left-to-right or right-to-left dilution. | |
7. Serial Dilution Transfer | 6 | Moves to the lowest concentration dilution in the serial dilution plate. • If a blowout volume is specified, aspirates an air gap. For details, see protocol step 4, above. • If specified, prewets the pipette tips in the designated wells. • Aspirates the sample into the pipette tips, and then dispenses the sample into the next set of dilution wells in the serial dilution plate. • If specified, mixes the well contents for the set number of cycles. The volume used for the mixing process is automatically calculated using the following formula. – If (x - 50 µL) > Tip Capacity, then Mixing Volume = Tip Capacity – If 50 µL ≤ x ≤ Syringe/Tip Capacity, then Mixing Volume = 0.75x – If x < 50 µL, then Mixing Volume = 0.5x where, x is the final volume in the destination wells, and maximum 250 µL Tip Capacity = 140 µL Default: 10 Range: 0–100 Note: – Generally, aqueous solutions are well mixed by 10 cycles if the maximum intermediate volume in the serial dilution (see step 4 in the setup tool) is > 200 µL. The intermediate serial dilution volumes are always higher than the final volume. More viscus and larger volume solutions require more mix cycles, which must be determined empirically. – The more mix cycles you use, the longer the run time. • If a blowout volume is specified, the head raises to near the top of the well, and the specified volume of air is dispensed followed by a tip touch on the east and west sides of the well. |
8. Optional Change Tips | 3 | If the change tips option is specified, ejects the used pipette tips into the tip box. If the change tips option is not specified, this step is skipped and the same set of pipette tips are used for all the serial dilution transfers. |
5 | Picks up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips in the seating station. • Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. • Picks up tips in column format for left-to-right or right-to-left dilution. | |
9. Additional Serial Dilution Transfers | 6 | Moves to the lowest concentration wells of the dilution series in the serial dilution plate. • If a blowout volume is specified, aspirates an air gap. • If specified, prewets the pipette tips with the sample. • Aspirates the sample into the pipette tips, and then dispenses the sample into the next set of dilution wells in the serial dilution plate. • If specified, mixes the well contents for the set number of cycles. • If specified, performs a blowout, followed by a tip touch on the east and west sides of the wells. • Repeats steps 7 and 8 until the serial dilution series is complete. |
10. Final Tip Ejection | 3 | Ejects the used pipette tips into the tip box. |
11. Park Head | 1 | Moves to a safe height above the wash station. |