Assay development guidelines
This topic provides guidelines for adjusting the protocol, and notes about the application.
Note: For maximum well capacities and recommended excess volumes in the source plate and destination plate, see the Labware Reference Guide in the Literature Library page of the workbench.
Automation movements and stepwise guidelines
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 of the tip-seating force that is used for this step. |
5 | Ejects the pipette tips into the seating station. | |
4. Single Tip Pickup | 5 | Positions the head at an offset from the seating station to use probe A12 to pick up the next available individual pipette tip, starting at H1. |
5. Prep for Transfer | 2 | Positions the head at an offset to the source plate so that the A12 pipette tip moves to the designated well. • 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 A12 pipette tip in the designated source well. Prewetting pipette tips is best practice for good pipetting accuracy and precision. Default: Yes Options: Yes, No • If specified, mixes the sample in the source well the specified number of cycles. The volume used for the mixing process is automatically calculated using the following formula. Mixing 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 |
Note: – The mix cycle default is set at 0 to ensure that mixing is not done unintentionally. If samples are centrifuged before reformatting, mixing would not be wanted. – Generally, aqueous solutions are well mixed by 5 cycles if the volume is <200 µL. 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.  Mix cycles are not recommended if any of the source wells will be used multiple times, as the mixing calculation assumes the volume in the source well is the Starting volume (µL) in source wells value. Therefore, after aspirating the initial volume, the mixing volume could be greater than the volume remaining in the well, and the mixing could introduce bubbles. The software assumes that all the wells in the source plate have the same volume. If any well has less volume than the volume set in the Starting volume (µL) in source wells field, mixing could introduce bubbles. | ||
6. Transfer | 2 | Aspirates the specified volume from the source plate well into the A12 pipette tip. |
6 | Positions the head at an offset to the destination plate so that the A12 pipette tip moves to the designated well. • Dispenses the sample into the destination well. • If specified, mixes the sample in the well the specified number of cycles. The volume used for the mixing process is automatically calculated using the following formula. Mixing 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 Note: – The mix cycle default is set at 0 to ensure that mixing is not done unintentionally, and is typically only done if there is a pre-existing volume in the destination well. – Generally, aqueous solutions are well mixed by 5 cycles if the volume is <200 µL. 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. The mixing volume may be far too low for destination wells that receive multiple transfers because the mixing volume calculation assumes that the volume in the destination well is the single transfer volume plus the pre-existing volume in the destination well. • 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. | |
7. Single Tip Eject | 3 | Ejects the used pipette tip into the tip box |
8. Additional Transfers | multiple | Repeats processes 3 through 6 for every designated well in the source plate. |
9. Park Head | 1 | Moves to a safe height above the wash station. |