ExperimentDigitalPCR
ExperimentDigitalPCR[Samples,PrimerPairs,Probes]⟹Protocol
creates a digital polymerase chain reaction Protocol, which uses a digital PCR instrument to quantify DNA or RNA targets by partitioning a mixture of Samples, PrimerPairs and fluorescent Probes into droplets, performing PCR and counting droplets with or without fluorescence signal.
ExperimentDigitalPCR[Samples]⟹Protocol
creates a digital polymerase chain reaction Protocol, which uses a digital PCR instrument to quantify DNA or RNA targets by partitioning reaction mixture provided in Samples into droplets, performing PCR and counting droplets with or without fluorescence signal.
Droplet Digital Polymerase Chain Reaction (ddPCR) is a method used for absolute quantification of DNA or RNA targets in samples. In a typical experiment, the samples are prepared with the addition of target sequence-specific primer pairs and probes, and the required enzymes, buffers and nucleotides. The samples are loaded into cartridges and partitioned into nanoliter-sized droplets using on-board microfluidic channels. Targets are amplified within the droplets using thermocycling, which leads to a release of reporter fluorophores from probes. Fluorescence amplitude signals are read from each droplet and the number of target copies are estimated using a ratio of droplets with and without fluorescence signal.
Experimental Principles
Figure 1.1: Step 1: Reaction mixtures are prepared and 20 μL of each reaction is transferred to a well of GCR96 cartridge using a liquid handler. Since 16-well blocks are processed for droplet generation in parallel, wells without samples in such a block are filled with PassiveWellBuffer. No data is generated from passive wells. Step 2: Cartridge is sealed with pierceable foil and load it into the input cartridge holder of the ddPCR instrument. Step 3: The instrument generates nanoliter-sized droplets from each input sample at a rate of 1000 droplets/μL of sample. Step 4: Polymerase chain reaction is carried out using a thermal cycler to amplify target genes. Step 5: Droplets are separated and read for fluorescence in 4 channels (emission wavelengths: 517 nm (FAM), 556 nm (HEX/VIC), 665 nm (Cy5) & 694 nm (Cy5.5)). Step 6: Data consisting of counts of positive or negative droplets can be used to quantify target concentration in samples and analyzed using AnalyzeDigitalPCR function.
Instrumentation
Bio-Rad QX One
Figure 2.1: Bio-Rad QX ONE is a fully automated droplet digital PCR instrument that is used for quantifying target DNA/RNA sequences. QX One contains an input cartridge holder that can house up to five assay cartridges. Once the cartridges are loaded and the protocol is set up, the instrument generates droplets from samples, conducts thermal cycling and detects fluorescence signal amplitudes from droplets. Each loaded and sealed assay cartridge contains a microfluidic unit that consists of a sample well and a droplet well. Oil and vacuum lines are used by the instrument to generate droplets for 16 wells in parallel. Once droplets are generated, the cartridge is moved to the thermal cycler, which executes the input thermal cycling protocol. The cartridge is then moved to the droplet reader, which separates the droplets by adding droplet reader oil. If a singularized droplet fits the size and shape requirements, it's fluorescence signal profile is recorded. After all the droplets are read, the cartridge is moved to the completed cartridge holder.
Experiment Options
General
Instrument
The device that takes a reaction mixture with target DNA or RNA, primers, probes and master mix, partitions them into nanoliter droplets, amplifies the nucleic acid templates, detects fluorescence signals from the droplets and quantifies the copy number of DNA or RNA targets.
Pattern Description: An object of type or subtype Model[Instrument, Thermocycler, DigitalPCR] or Object[Instrument, Thermocycler, DigitalPCR]
Programmatic Pattern: ObjectP[{Model[Instrument, Thermocycler, DigitalPCR], Object[Instrument, Thermocycler, DigitalPCR]}]
PreparedPlate
Indicates if the input with samples, primers, probes and master mix is already loaded on a DropletCartridge and ready to perform digital PCR without further reagent addition.
Default Calculation: Automatically resolves to True if the input samples are in a container matching "Model[Container,Plate,DropletCartridge,"Bio-Rad GCR96 Digital PCR Cartridge"]", and no PrimerPair and Probe inputs are specified, otherwise resolves to False.
DropletCartridge
The plate with integrated microfluidics used to partition samples into droplets. Each microfluidic unit contains a sample well, channels for the sample, oil and vacuum lines to facilitate the formation of nanoliter size droplets, and a well to collect the generated droplets.
Pattern Description: An object of type or subtype Model[Container, Plate, DropletCartridge] or list of one or more an object of type or subtype Object[Container, Plate, DropletCartridge] or a prepared sample entries.
Programmatic Pattern: ObjectP[Model[Container, Plate, DropletCartridge]] | {(ObjectP[Object[Container, Plate, DropletCartridge]] | _String)..}
DropletGeneratorOil
The immiscible liquid that will be used to generate nanoliter-sized aqueous droplets in microfluidic channels of DropletCartridge.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample.
DropletReaderOil
The immiscible liquid that will be used to separate PCR amplified droplets and facilitate fluorescence signal detection from individual droplets.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample.
AmplitudeMultiplexing
Number of genetic targets to be detected in each fluorescence channel. Multiple genetic targets can be detected in a single channel using gradations of different primer and probe concentrations to control amplification of the gene and fluorescence signal amplitude of droplets.
Figure 3.1: Amplitude multiplexing with Bio-Rad QX One. In addition to multiplexing targets in the 4 available channels, amplitude multiplexing allows for quantifying 8 or more targets in a sample by detecting more than 1 target in each channel. Since target quantification with DigitalPCR is performed by counts of negative and positive droplets, sufficient separation of droplet populations is required. For amplitude multiplexing, separation of droplet populations can be achieved by controlling the concentration of primer pairs and probes. In this example, targets 1 and 2 are detected in Channel 1, and targets 3 and 4 are detected in Channel 2. Left: Sample composition contains 2x higher concentration for targets 1 and 3 compared to targets 2 and 4. Right: Differences in concentrations of primers and probes lead to corresponding differences in signal amplitudes from droplets with target 1 or 3 compared to those with target 2 or 4. Additional droplet populations with higher signal amplitudes as well as signals in Channels 1 and 2 results from various combinations of targets 1, 2, 3 and 4. Analysis of target copy number can be performed once droplet populations with all possible target combinations are isolated.
Default Calculation: Automatically tallies the number of amplification targets in each fluorescence channel using the fluorophore emission wavelength of target probe(s) and reference probe(s). If any channel has more than one target and/or reference, resolves to a list of number of targets per channel; otherwise resolves to Null.
NumberOfReplicates
Number of times each of the input samples should be analyzed using identical experimental parameters.
Sample Preparation
SampleVolume
For each sample, the portion of the reaction volume that is made up of the sample from which the target is amplified.
Default Calculation: Automatically resolves to 0μL when PreparedPlate is True, otherwise resolves to 4μL.
Pattern Description: Greater than or equal to 0 microliters and less than or equal to 100 microliters.
PremixedPrimerProbe
For each sample, indicates if primerPair and probe input has an assay that contains a set of primers and a probe or if a primerPair input contains a primer set or if all three index-matched inputs are separate objects. The primerPair and probe inputs should have the same object of type Object[Sample] or Model[Sample]. Volume of the pre-mixed assay is calculated from the concentration of the probe oligomer in the mixture.
Default Calculation: Automatically resolves to TargetAssay if the primerPair and probe inputs have the same object, resolves to PrimerSet if only primerPair inputs have the same object, resolves to None if primerPair and probe have unique objects and resolves to Null if primerPairs and probes inputs are Null.
ForwardPrimerConcentration
For each sample, the molarity of the short oligomer designed to bind the antisense strand of a template sequence. A primer functions as an anchor for polymerases and transcriptases during polymerase chain reaction.
Default Calculation: Automatically resolves to match ReversePrimerConcentration; otherwise calculated using the formula: ForwardPrimerConcentration=(max concentration)*Table[(N-t)/N,{t,0,T-1}], where (max concentration)=1350 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); or resolves to 900 nM for forward primer in channels with a single amplification product.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 1500 nanomolar entries or Null.
ForwardPrimerVolume
Default Calculation: Automatically calculated using the formula: ForwardPrimerVolume=(ForwardPrimerConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
ReversePrimerConcentration
For each sample, the molarity of the short oligomer designed to bind the sense (coding) strand of a template sequence. A primer functions as an anchor for polymerases and transcriptases during polymerase chain reaction.
Default Calculation: Automatically resolves to match ForwardPrimerConcentration; otherwise calculated using the formula: ReversePrimerConcentration=(max concentration)*Table[(N-t)/N,{t,0,T-1}], where (max concentration)=1350 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); or resolves to 900 nM for reverse primer in channels with a single amplification product.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 1500 nanomolar entries or Null.
ReversePrimerVolume
Default Calculation: Automatically calculated using the formula: ReversePrimerConcentration=(ReversePrimerConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
ProbeConcentration
For each sample, the molarity of the short oligomer strand containing a reporter and quencher, and designed to be complementary to a template sequence being amplified. Fluorescence signal is observed when the probe is hydrolyzed by a polymerase and the fluorophore is released.
Default Calculation: Automatically calculated using the formula: ProbeConcentration=(max concentration)*Table[(N-t)/N,{t,0,T-1}], where (max concentration)=375 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); otherwise resolves to 250 nM for reverse primer in channels with a single amplification product.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 500 nanomolar entries or Null.
ProbeVolume
Default Calculation: Automatically resolved using the formula: ProbeVolume=(ProbeConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
ReferencePrimerPairs
For each sample, the pair of short oligomer strands designed to amplify a reference gene (such as a housekeeping gene), whose expression should not be different between samples. Primers can be specified as individual objects for forward and reverse primers. Primer-sets and pre-mixed assays with primers and probes can also be used.
Pattern Description: List of one or more {Reference Forward Primer, Reference Reverse Primer} entries or {Reference Forward Primer, Reference Reverse Primer} or Null.
Programmatic Pattern: ({{ObjectP[{Model[Sample], Object[Sample]}] | _String, ObjectP[{Model[Sample], Object[Sample]}] | _String}..} | {Null, Null}) | Null
ReferencePremixedPrimerProbe
For each sample, indicates if ReferencePrimerPairs and ReferenceProbes have assays that contains a set of primers and a probe or if a ReferencePrimerPairs input contains a primer set or if all three index-matched reference objects are separate. The ReferencePrimerPairs and ReferenceProbes should have the same object of type Object[Sample] or Model[Sample]. Volume of the pre-mixed assay is calculated from the concentration of the probe oligomer in the mixture.
Default Calculation: Automatically resolves to TargetAssay if the reference primer pair and reference probe inputs have the same object, resolves to PrimerSet if only reference primer pair inputs have the same object, resolves to None if reference primer pair and reference probe have unique objects and resolves to Null if reference primer pairs and probes are Null.
ReferenceForwardPrimerConcentration
For each sample, the molarity of reference gene(s) specific oligomer that binds to the antisense strand of the template.
Default Calculation: Automatically resolves to match ReferenceReversePrimerConcentration; otherwise calculated using the formula: ReferenceForwardPrimerConcentration=(max concentration)*Table[(N-t)/N,{t,T,N-1}], where (max concentration)=1350 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); or resolves to 900 nM for forward primer in channels with a single amplification product or Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 1500 nanomolar entries or Null.
ReferenceForwardPrimerVolume
For each sample, the volume of forward primer(s) for reference gene(s) that will be added to the reaction.
Default Calculation: Automatically calculated using the formula: ReferenceForwardPrimerConcentration=(ReferenceForwardPrimerConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object; Resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
ReferenceReversePrimerConcentration
For each sample, the molarity of reference gene(s) specific oligomer that binds to the sense (coding) strand of the template.
Default Calculation: Automatically resolves to match ReferenceForwardPrimerConcentration; otherwise calculated using the formula: ReferenceReversePrimerConcentration=(max concentration)*Table[(N-t)/N,{t,T,N-1}], where (max concentration)=1350 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); or resolves to 900 nM for reverse primer in channels with a single amplification product or Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 1500 nanomolar entries or Null.
ReferenceReversePrimerVolume
For each sample, the volume of reverse primer(s) for reference gene(s) that will be added to the reaction.
Default Calculation: Automatically calculated using the formula: ReferenceReversePrimerConcentration=(ReferenceReversePrimerConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object; Resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
ReferenceProbes
For each sample, the short oligomer strand containing a fluorescence reporter and quencher, which enhances its fluorescence upon separation from the quencher.
Pattern Description: List of one or more an object of type or subtype Model[Sample] or Object[Sample] or a prepared sample entries or Null.
ReferenceProbeConcentration
Default Calculation: Automatically calculated using the formula: ReferenceProbeConcentration=(max concentration)*Table[(N-t)/N,{t,T,N-1}], where (max concentration)=375 Nanomolar, N=(number of targets + number of references) in a channel resolved in AmplitudeMultiplexing, and T=(number of targets); otherwise resolves to 250 nM for probes in channels with a single amplification product or Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 nanomolar and less than or equal to 500 nanomolar entries or Null.
ReferenceProbeVolume
For each sample, the volume of the probe(s) for reference gene(s) that will be added to the reaction.
Default Calculation: Automatically resolved using the formula: ProbeVolume=(ProbeConcentration*ReactionVolume)/(stock concentration), where stock concentration is downloaded from the compositional information of the corresponding oligomer object; resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more greater than or equal to 0 microliters and less than or equal to 100 microliters entries or Null.
MasterMix
For each sample, the stock solution composed of enzymes (DNA polymerase and optionally reverse transcriptase), buffer and nucleotides used to amplify DNA or RNA targets in the sample. It is recommended to select the MasterMix even if it is in the sample as the type of master mix can affect the size of droplets generated and selection of droplets for signal detection.
Default Calculation: Automatically resolves to Model[Sample,"Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes, Buffer"] if input samples contain RNA templates or if any reverse transcription options are informed. The RT buffer requires addition of reverse transcriptase and DTT, so the master mix will be automatically replaced with Model[Sample,StockSolution,"Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes, 2x Master Mix"] in the MasterMixes field of Object[Protocol,DigitalPCR]. Resolves to Model[Sample,"Bio-Rad ddPCR Multiplex Supermix"] if multiple probes are used, otherwise resolves to Model[Sample,"Bio-Rad ddPCR Supermix for Probes (No dUTP)"].
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample.
MasterMixConcentrationFactor
For each sample, the amount by which the MasterMix stock solution must be diluted with Diluent in order to achieve 1X buffer concentration in the reaction.
Default Calculation: Automatically resolves to Null if PreparedPlate is True, otherwise set to ConcentratedBufferDilutionFactor from Model[Sample] of MasterMix.
MasterMixVolume
For each sample, the volume of the stock solution (composed of enzymes (DNA polymerase and optionally reverse transcriptase), buffer and nucleotides) that will be added to the sample.
Default Calculation: Automatically resolves to Null if PreparedPlate is True, otherwise set to MasterMixVolume=ReactionVolume/MasterMixConcentrationFactor.
Pattern Description: Greater than or equal to 0 microliters and less than or equal to 100 microliters or Null.
Diluent
For each sample, stock solution used to reach ReactionVolume, once all other components have been added.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample.
DiluentVolume
Default Calculation: Automatically set according to the equation DiluentVolume=ReactionVolume-(SampleVolume+ForwardPrimerVolume+ReversePrimerVolume+ProbeVolume+ReferenceForwardPrimerVolume+ReferenceReversePrimerVolume+ReferenceProbeVolume+MasterMixVolume) if Diluent is not set to Null, or Null otherwise.
Pattern Description: Greater than or equal to 0 microliters and less than or equal to 100 microliters or Null.
ReactionVolume
For each sample, the volume of reaction including sample, primers, probes, master mix and buffer. A LoadingVolume of 21 Microliter will be transferred to DropletCartridge well after reaction components are mixed.
Pattern Description: Greater than or equal to 30 microliters and less than or equal to 100 microliters.
ActiveWell
The well in which the reaction will be conducted. The position can be designated with a well index when a single DropletCartridge is in use. An alternate input with plate index and well index can be used to configure multiple DropletCartridge plates in a single experiment. Samples can be distributed in up to 5 individual 96-well DropletCartridges.
Default Calculation: Automatically resolves the well indices. For variable thermocycling conditions, samples are grouped with compatible conditions. Samples containing RNA are prioritized for droplet generation and thermocycling.
Pattern Description: List of one or more a string that matches the pattern: WellP entries or list of one or more {Plate Index, Well Index} entries.
PassiveWells
Droplet generation is conducted in parallel on a section of 16 sample units in a DropletCartridge. Each section is composed of an adjacent pair of an odd and an even numbered column such as {1,2}. Unused wells in a section are filled with viscosity matched control buffer to enable parallel processing and no data is generated from these wells.
Default Calculation: Automatically resolves the well indices for empty wells within a 16 unit section that has 1 or more samples. Resolves to None if each 16 unit section in use is filled with samples. Sections without any samples are skipped during the assay.
Pattern Description: List of one or more a string that matches the pattern: WellP entries or list of one or more {Plate Index, Well Index} entries or None.
PassiveWellBuffer
Control solution with viscosity matched to master mix. 20 Microliter of buffer is added to PassiveWells.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample.
Sample Dilution
SampleDilution
For each sample, indicates if Diluent should be used to generate a series of decreasing target concentrations to ensure that an unknown sample falls within the DigitalPCR detection range of 0.25 to 5,000 targets/μL.
SerialDilutionCurve
For each sample, the collection of dilutions that are performed before the SampleVolume is transferred to a mixture of primers, probes, MasterMix and Diluent. For Serial Dilution Factors, the sample will be diluted with the Diluent by the dilution factor at each transfer step. For example, a SerialDilutionCurve of {36*Microliter,{1,0.8,0.5,0.5}} for a 100 mg/mL sample will result in 4 dilutions with concentrations of 100 mg/mL, 80 mg/mL, 40 mg/mL, and 20 mg/mL. For Serial Dilution Volumes, the Transfer Volume is taken out of the sample and added to a second well with the Diluent Volume of the Diluent. It is mixed, then the Transfer Volume is taken out of that well to be added to a third well. This is repeated to make Number Of Dilutions diluted samples. For example, if a 100 mg/mL sample with a Transfer Volume of 30 Microliters, a Diluent Volume of 10 Microliters and a Number of Dilutions of 2 is used, it will create a DilutionCurve of 75 mg/mL, 56.3 mg/mL, and 42.2 mg/mL.
Figure 3.2: Use the SerialDilutionCuve option to create a collection of serial dilutions that will be performed on the sample.
Default Calculation: Automatically resolves to {50*Microliter,{0.1,4}}, where 50μL is the sample volume, 0.1 is the constant dilution factor and 4 is the number of dilutions, when SampleDilution is True, otherwise resolves to Null.
Programmatic Pattern: (({GreaterP[0*Microliter], GreaterEqualP[0*Microliter], GreaterP[1, 1]} | {GreaterEqualP[10*Microliter], {RangeP[0, 1], GreaterP[1, 1]} | {RangeP[0, 1]..}}) | Automatic) | Null
DilutionMixVolume
Default Calculation: Automatically resolves to 10 μL if SampleDilution is True, otherwise resolves to Null.
DilutionNumberOfMixes
For each sample, the number of cycles of pipetting in and out that is used to mix the sample and the Diluent.
Default Calculation: Automatically resolves to 10 if SampleDilution is True, otherwise resolves to Null.
Pattern Description: Greater than or equal to 0 and less than or equal to 20 in increments of 1 or Null.
DilutionMixRate
For each sample, the speed at which the DilutionMixVolume is pipetted in and out to mix the sample and the Diluent.
Default Calculation: Automatically resolves to 30μL/s if SampleDilution is True, otherwise resolves to Null.
Pattern Description: Greater than or equal to 0.4 microliters per second and less than or equal to 250 microliters per second or Null.
Plate Seal
PlateSealInstrument
Pattern Description: An object of type or subtype Model[Instrument, PlateSealer] or Object[Instrument, PlateSealer]
PlateSealFoil
PlateSealTemperature
Pattern Description: Greater than or equal to 100 degrees Celsius and less than or equal to 190 degrees Celsius.
PlateSealTime
Reverse Transcription
ReverseTranscription
For each sample, indicates if one-step reverse transcription is performed in order to convert RNA input samples to cDNA. Thermocycling is performed after samples are partitioned into droplets and collected in the droplet well of their corresponding microfluidic unit. All sample emulsions on a DropletCartridge are thermocycled simultaneously.
Default Calculation: Automatically set to True if any reverse transcription related options are set, and False if none are set.
ReverseTranscriptionTime
For each sample, the length of time for which the reaction volume will held at ReverseTranscriptionTemperature in order to convert RNA to cDNA.
Default Calculation: Automatically set to 15 minutes if ReverseTranscription is set to True, and Null if it is set to False.
ReverseTranscriptionTemperature
For each sample, the initial temperature at which the sample should be heated to in order to activate the reverse transcription enzymes in the master mix to convert RNA to cDNA.
Default Calculation: Automatically set to 50°Celsius if ReverseTranscription is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
ReverseTranscriptionRampRate
For each sample, the rate at which the reaction volume is heated to bring the sample to the reverse transcription temperature.
Default Calculation: Automatically set to 1.6°Celsius/Second if ReverseTranscription is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second or Null.
Polymerase Activation
Activation
For each sample, indicates if polymerase hot start activation is performed. In order to reduce non-specific amplification, modern enzymes can be made room temperature stable by inhibiting their activity via thermolabile conjugates. Once an experiment is ready to be run, this inhibition is removed by heating the reaction to ActivationTemperature. The activation step is recommended as ActivationTemperature promotes stabilization of droplet boundary through a reaction between MasterMix and DropletGeneratorOil prior to thermocycling.
ActivationTime
For each sample, the length of time for which the reaction volume is held at ActivationTemperature in order to remove the thermolabile conjugates inhibiting enzyme activity.
ActivationTemperature
For each sample, the temperature at which at which the thermolabile conjugates inhibiting enzyme activity are removed.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
ActivationRampRate
For each sample, the rate at which the reaction is heated to bring the sample to the ActivationTemperature.
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second or Null.
Denaturation
DenaturationTime
For each sample, the length of time for which the reaction is held at DenaturationTemperature in order to separate any double stranded template DNA into single strands.
DenaturationTemperature
For each sample, the temperature to which the sample is heated in order to disassociate double stranded template DNA into single strands.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 100 degrees Celsius.
DenaturationRampRate
For each sample, the rate at which the reaction is heated to bring the sample to DenaturationTemperature.
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second.
Primer Annealing
PrimerAnnealing
For each sample, indicates if annealing should be performed. Lowering the temperature during annealing allows primers to bind to template DNA to serve as anchor points for DNA polymerization. It is highly recommended to use PrimerAnnealing as a single step to perform annealing and extension if the working temperature of the polymerase in MasterMix is 60°Celsius.
PrimerAnnealingTime
For each sample, the length of time for which the reaction is held at PrimerAnnealingTemperature in order to allow binding of primers to the template DNA.
Default Calculation: Automatically set to 60 seconds if PrimerAnnealing or PrimerGradientAnnealing is set to True, and Null if it is set to False.
PrimerAnnealingTemperature
For each sample, the temperature to which the sample is heated in order to allow primers to bind to the template strands.
Default Calculation: Automatically set to 60°Celsius if PrimerAnnealing is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
PrimerAnnealingRampRate
For each sample, the rate at which the reaction is heated to bring the sample to PrimerAnnealingTemperature.
Default Calculation: Automatically set to 2.0°Celsius/Second if PrimerAnnealing is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second or Null.
Primer Gradient Annealing
PrimerGradientAnnealing
For each sample, indicates if a gradation of temperature should be used for annealing across columns such that each row has the same annealing temperature. A linearly decreasing series of temperatures for 8 rows is calculated as follows: Range[Tmin,Tmax,(Tmax-Tmin)/7], where Tmin is the minimum temperature set to Row 'A' and Tmax is the maximum temperature set to Row 'H'. A temperature gradient can be used to optimize annealing by setting Tmin and Tmax to be (expected annealing temperature) +/- 5 Celsius. An aliquot of reaction mixture with the same target, primer pair and probe is added to at least one well per row of a DropletCartridge and the amplification efficiency is determined by the fluorescence signal amplitude of the droplets after PCR.
PrimerGradientAnnealingMinTemperature
For each sample, the lower value of the temperature range that will be used to calculate annealing temperature of each row.
Default Calculation: Automatically set to 55°Celsius if PrimerGradientAnnealing is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 30 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
PrimerGradientAnnealingMaxTemperature
For each sample, the upper value of the temperature range that will be used to calculate annealing temperature of each row.
Default Calculation: Automatically set to 65°Celsius if PrimerGradientAnnealing is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 30 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
PrimerGradientAnnealingRow
For each sample, the tier in which the sample will be located in the DropletCartridge. "Row A" is the minimum temperature and "Row H" is the maximum temperature, while the rows in between have a gradation of temperature.
Default Calculation: Automatically resolves the row & temperature assignment for sample with PrimerGradientAnnealing set to True, where the first sample is assigned to "Row A", second sample to "Row B" and so on; otherwise resolves to Null if PrimerGradientAnnealing is False.
Pattern Description: Row A, Row B, Row C, Row D, Row E, Row F, Row G, or Row H or {Row, Temperature} or Null.
Programmatic Pattern: ((("Row A" | "Row B" | "Row C" | "Row D" | "Row E" | "Row F" | "Row G" | "Row H") | {"Row A" | "Row B" | "Row C" | "Row D" | "Row E" | "Row F" | "Row G" | "Row H", RangeP[30*Celsius, 100*Celsius]}) | Automatic) | Null
Strand Extension
Extension
ExtensionTime
For each sample, the length of time for which the polymerase synthesizes a new DNA strand using the provided primer pairs and template DNA.
Default Calculation: Automatically set to 60 Second if Extension is set to True, and Null if it is set to False.
ExtensionTemperature
For each sample, the temperature at which the sample is held to allow the polymerase to synthesis new DNA strand from the template DNA.
Default Calculation: Automatically set to 60°Celsius if Extension is set to True, and Null if it is set to False.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 100 degrees Celsius or Null.
ExtensionRampRate
For each sample, the rate at which the reaction is heated to bring the sample to ExtensionTemperature.
Default Calculation: Automatically set to 2.0°Celsius/Second if Extension is set to True, and Null if it is set to False
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second or Null.
NumberOfCycles
For each sample, the remaining number of times the samples will undergo repeated rounds of denaturation, annealing and extension to amplify targets.
Polymerase Degradation
PolymeraseDegradation
For each sample, indicates if the polymerase should be degraded at PolymeraseDegradationTemperature. After thermocycling is complete, the DropletCartridge is will remain at 25 Celsius (i.e. ambient temperature). Polymerase degradation is recommended to prevent any alterations to the droplet contents prior to signal detection.
PolymeraseDegradationTime
For each sample, the length of time for which the sample is held at PolymeraseDegradationTemperature.
Default Calculation: Automatically set to 10 minutes if PolymeraseDegradation is set to True, or Null otherwise
PolymeraseDegradationTemperature
Default Calculation: Automatically set to 98 Celsius if PolymeraseDegradation is set to True, or Null otherwise.
Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
PolymeraseDegradationRampRate
Default Calculation: Automatically set to 2.5 degrees Celsius per second if PolymeraseDegradation is set to True, or Null otherwise.
Pattern Description: Greater than or equal to 1. degrees Celsius per second and less than or equal to 2.5 degrees Celsius per second or Null.
Detection
ProbeFluorophore
For each sample, the fluorescent molecule conjugated to the probe that allows for detection of amplification.
ProbeExcitationWavelength
Default Calculation: Automatically set to FluorescenceExcitationMaximums of the fluorophore attached to the probe.
Pattern Description: List of one or more 495. nanometers, 535. nanometers, 538. nanometers, 647. nanometers, or 675. nanometers entries.
ProbeEmissionWavelength
Default Calculation: Automatically set to FluorescenceEmissionMaximums of the fluorophore attached to the probe.
Pattern Description: List of one or more 517. nanometers, 554. nanometers, 556. nanometers, 665. nanometers, or 694. nanometers entries.
ReferenceProbeFluorophore
The fluorescent molecule conjugated to the reference probe that allows for detection of amplification.
Default Calculation: Automatically set to the model of fluorophore attached to the reference probe oligomer; resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more an object of type or subtype Model[Molecule] entries or Null.
ReferenceProbeExcitationWavelength
Default Calculation: Automatically set to FluorescenceExcitationMaximums of the fluorophore attached to the reference probe; resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more 495. nanometers, 535. nanometers, 538. nanometers, 647. nanometers, or 675. nanometers entries or Null.
ReferenceProbeEmissionWavelength
The wavelength of light emitted by the reporter once it has been excited and read by the instrument.
Default Calculation: Automatically set to FluorescenceEmissionMaximums of the fluorophore attached to the reference probe; resolves to Null if ReferencePrimerPairs is Null.
Pattern Description: List of one or more 517. nanometers, 554. nanometers, 556. nanometers, 665. nanometers, or 694. nanometers entries or Null.
Post Experiment
ForwardPrimerStorageCondition
For each sample, the non-default conditions under which the forward primers of this experiment should be stored after the protocol is completed. If left unset, the forward primers will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
ReversePrimerStorageCondition
For each sample, the non-default conditions under which the reverse primers of this experiment should be stored after the protocol is completed. If left unset, the reverse primers will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
ProbeStorageCondition
For each sample, the non-default conditions under which the probes of this experiment should be stored after the protocol is completed. If left unset, the probes will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
ReferenceForwardPrimerStorageCondition
For each sample, the non-default conditions under which the forward primers for the reference of this experiment should be stored after the protocol is completed. If left unset, the forward primers for the reference will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
ReferenceReversePrimerStorageCondition
For each sample, the non-default conditions under which the reverse primers for the reference of this experiment should be stored after the protocol is completed. If left unset, the reverse primers for the reference will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
ReferenceProbeStorageCondition
For each sample, the non-default conditions under which the probes for the reference of this experiment should be stored after the protocol is completed. If left unset, the probes for the reference will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
MasterMixStorageCondition
For each sample, the non-default condition under which MasterMix of this experiment should be stored after the protocol is completed. If left unset, MasterMix will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
SamplesInStorageCondition
The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
Model Input
PreparedModelContainer
Indicates the container in which a Model[Sample] specified as input to the experiment function will be prepared.
Default Calculation: If PreparedModelAmount is set to All and when the input model has a product associated with both Amount and DefaultContainerModel populated, automatically set to the DefaultContainerModel value in the product. Otherwise set to Model[Container, Vessel, "2mL Tube"].
PreparedModelAmount
Indicates the amount of a Model[Sample] specified as input to the experiment function that will be prepared in the PreparedModelContainer. When set to All and the input model sample is not preparable, the entire amount of the input model sample that comes from one of the Products is prepared. The selected product must have both Amount and DefaultContainerModel populated, and it must not be a KitProduct. When set to All and the input model is preparable such as water, 1 Milliliter of the input model sample is prepared.
Sample Prep Options
Sample Preparation
PreparatoryUnitOperations
Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSamplePreparation.
Pattern Description: List of one or more unit Operation ManualSamplePreparation or RoboticSamplePreparation or unit Operation must match SamplePreparationP entries or Null.
Programmatic Pattern: {((ManualSamplePreparationMethodP | RoboticSamplePreparationMethodP) | SamplePreparationP)..} | Null
Preparatory Incubation
Incubate
Indicates if the SamplesIn should be incubated at a fixed temperature prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Incubation options are set. Otherwise, resolves to False.
IncubationTemperature
Temperature at which the SamplesIn should be incubated for the duration of the IncubationTime prior to starting the experiment.
Pattern Description: Ambient or greater than or equal to -20 degrees Celsius and less than or equal to 500 degrees Celsius or Null.
Programmatic Pattern: ((Ambient | RangeP[$MinIncubationTemperature, $MaxIncubationTemperature]) | Automatic) | Null
IncubationTime
Duration for which SamplesIn should be incubated at the IncubationTemperature, prior to starting the experiment.
Mix
Default Calculation: Automatically resolves to True if any Mix related options are set. Otherwise, resolves to False.
MixType
Default Calculation: Automatically resolves based on the container of the sample and the Mix option.
Pattern Description: Roll, Vortex, Sonicate, Pipette, Invert, Stir, Shake, Homogenize, Swirl, Disrupt, or Nutate or Null.
MixUntilDissolved
Indicates if the mix should be continued up to the MaxIncubationTime or MaxNumberOfMixes (chosen according to the mix Type), in an attempt dissolve any solute. Any mixing/incubation will occur prior to starting the experiment.
Default Calculation: Automatically resolves to True if MaxIncubationTime or MaxNumberOfMixes is set.
MaxIncubationTime
Maximum duration of time for which the samples will be mixed while incubated in an attempt to dissolve any solute, if the MixUntilDissolved option is chosen. This occurs prior to starting the experiment.
Default Calculation: Automatically resolves based on MixType, MixUntilDissolved, and the container of the given sample.
IncubationInstrument
Default Calculation: Automatically resolves based on the options Mix, Temperature, MixType and container of the sample.
Pattern Description: An object of type or subtype Model[Instrument, Roller], Model[Instrument, OverheadStirrer], Model[Instrument, Vortex], Model[Instrument, Shaker], Model[Instrument, BottleRoller], Model[Instrument, Roller], Model[Instrument, Sonicator], Model[Instrument, HeatBlock], Model[Instrument, Homogenizer], Model[Instrument, Disruptor], Model[Instrument, Nutator], Model[Instrument, Thermocycler], Model[Instrument, EnvironmentalChamber], Model[Instrument, Pipette], Object[Instrument, Roller], Object[Instrument, OverheadStirrer], Object[Instrument, Vortex], Object[Instrument, Shaker], Object[Instrument, BottleRoller], Object[Instrument, Roller], Object[Instrument, Sonicator], Object[Instrument, HeatBlock], Object[Instrument, Homogenizer], Object[Instrument, Disruptor], Object[Instrument, Nutator], Object[Instrument, Thermocycler], Object[Instrument, EnvironmentalChamber], or Object[Instrument, Pipette] or Null.
AnnealingTime
Minimum duration for which the SamplesIn should remain in the incubator allowing the system to settle to room temperature after the IncubationTime has passed but prior to starting the experiment.
IncubateAliquotContainer
The desired type of container that should be used to prepare and house the incubation samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
IncubateAliquotDestinationWell
The desired position in the corresponding IncubateAliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
IncubateAliquot
The amount of each sample that should be transferred from the SamplesIn into the IncubateAliquotContainer when performing an aliquot before incubation.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
Preparatory Centrifugation
Centrifuge
Indicates if the SamplesIn should be centrifuged prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Centrifuge options are set. Otherwise, resolves to False.
CentrifugeInstrument
Pattern Description: An object of type or subtype Model[Instrument, Centrifuge] or Object[Instrument, Centrifuge] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, Centrifuge], Object[Instrument, Centrifuge]}] | Automatic) | Null
CentrifugeIntensity
The rotational speed or the force that will be applied to the samples by centrifugation prior to starting the experiment.
Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
CentrifugeTime
CentrifugeTemperature
The temperature at which the centrifuge chamber should be held while the samples are being centrifuged prior to starting the experiment.
Pattern Description: Ambient or greater than or equal to -10 degrees Celsius and less than or equal to 40 degrees Celsius or Null.
CentrifugeAliquotContainer
The desired type of container that should be used to prepare and house the centrifuge samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
CentrifugeAliquotDestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
CentrifugeAliquot
The amount of each sample that should be transferred from the SamplesIn into the CentrifugeAliquotContainer when performing an aliquot before centrifugation.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
Preparatory Filtering
Filtration
Indicates if the SamplesIn should be filter prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Filter options are set. Otherwise, resolves to False.
FiltrationType
Default Calculation: Will automatically resolve to a filtration type appropriate for the volume of sample being filtered.
FilterInstrument
Default Calculation: Will automatically resolved to an instrument appropriate for the filtration type.
Pattern Description: An object of type or subtype Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], or Object[Instrument, SyringePump] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], Object[Instrument, SyringePump]}] | Automatic) | Null
Filter
The filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Will automatically resolve to a filter appropriate for the filtration type and instrument.
Pattern Description: An object of type or subtype Model[Container, Plate, Filter], Model[Container, Vessel, Filter], or Model[Item, Filter] or Null.
Programmatic Pattern: (ObjectP[{Model[Container, Plate, Filter], Model[Container, Vessel, Filter], Model[Item, Filter]}] | Automatic) | Null
FilterMaterial
The membrane material of the filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Resolves to an appropriate filter material for the given sample is Filtration is set to True.
Pattern Description: Cellulose, Cotton, Polyethylene, Polypropylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, AgaroseResin, CobaltResin, Silica, HLB, or AnoporeAlumina or Null.
PrefilterMaterial
The material from which the prefilter filtration membrane should be made of to remove impurities from the SamplesIn prior to starting the experiment.
Pattern Description: Cellulose, Cotton, Polyethylene, Polypropylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, AgaroseResin, CobaltResin, Silica, HLB, or AnoporeAlumina or Null.
FilterPoreSize
The pore size of the filter that should be used when removing impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Resolves to an appropriate filter pore size for the given sample is Filtration is set to True.
Pattern Description: 0.008 micrometers, 0.02 micrometers, 0.1 micrometers, 0.2 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
PrefilterPoreSize
The pore size of the filter; all particles larger than this should be removed during the filtration.
Pattern Description: 0.008 micrometers, 0.02 micrometers, 0.1 micrometers, 0.2 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
FilterSyringe
Default Calculation: Resolves to an syringe appropriate to the volume of sample being filtered, if Filtration is set to True.
Pattern Description: An object of type or subtype Model[Container, Syringe] or Object[Container, Syringe] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Container, Syringe], Object[Container, Syringe]}] | _String) | Automatic) | Null
FilterHousing
The filter housing that should be used to hold the filter membrane when filtration is performed using a standalone filter membrane.
Default Calculation: Resolve to an housing capable of holding the size of the membrane being used, if filter with Membrane FilterType is being used and Filtration is set to True.
Pattern Description: An object of type or subtype Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], or Object[Instrument, FilterBlock] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], Object[Instrument, FilterBlock]}] | Automatic) | Null
FilterIntensity
Default Calculation: Will automatically resolve to 2000 GravitationalAcceleration if FiltrationType is Centrifuge and Filtration is True.
Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
FilterTime
Default Calculation: Will automatically resolve to 5 Minute if FiltrationType is Centrifuge and Filtration is True.
FilterTemperature
The temperature at which the centrifuge chamber will be held while the samples are being centrifuged during filtration.
Default Calculation: Will automatically resolve to 22 Celsius if FiltrationType is Centrifuge and Filtration is True.
FilterContainerOut
The desired container filtered samples should be produced in or transferred into by the end of filtration, with indices indicating grouping of samples in the same plates, if desired.
Default Calculation: Automatically set as the PreferredContainer for the Volume of the sample. For plates, attempts to fill all wells of a single plate with the same model before using another one.
Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or {Index, Container} or Null.
Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | {GreaterEqualP[1, 1] | Automatic, (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
FilterAliquotDestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
FilterAliquotContainer
The desired type of container that should be used to prepare and house the filter samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
FilterAliquot
The amount of each sample that should be transferred from the SamplesIn into the FilterAliquotContainer when performing an aliquot before filtration.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
FilterSterile
Default Calculation: Resolve to False if Filtration is indicated. If sterile filtration is desired, this option must manually be set to True.
Aliquoting
Aliquot
Indicates if aliquots should be taken from the SamplesIn and transferred into new AliquotSamples used in lieu of the SamplesIn for the experiment. Note that if NumberOfReplicates is specified this indicates that the input samples will also be aliquoted that number of times. Note that Aliquoting (if specified) occurs after any Sample Preparation (if specified).
AliquotAmount
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container if a liquid, or the current Mass or Count if a solid or counted item, respectively.
Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | RangeP[1*Milligram, 20*Kilogram] | GreaterP[0*Unit, 1*Unit] | GreaterP[0., 1.] | All) | Automatic) | Null
TargetConcentration
The desired final concentration of analyte in the AliquotSamples after dilution of aliquots of SamplesIn with the ConcentratedBuffer and BufferDiluent which should be used in lieu of the SamplesIn for the experiment.
TargetConcentrationAnalyte
Default Calculation: Automatically set to the first value in the Analytes field of the input sample, or, if not populated, to the first analyte in the Composition field of the input sample, or if none exist, the first identity model of any kind in the Composition field.
Pattern Description: An object of type or subtype Model[Molecule], Model[Molecule, cDNA], Model[Molecule, Oligomer], Model[Molecule, Transcript], Model[Molecule, Protein], Model[Molecule, Protein, Antibody], Model[Molecule, Carbohydrate], Model[Molecule, Polymer], Model[Resin], Model[Resin, SolidPhaseSupport], Model[Lysate], Model[ProprietaryFormulation], Model[Virus], Model[Cell], Model[Cell, Mammalian], Model[Cell, Bacteria], Model[Cell, Yeast], Model[Tissue], Model[Material], or Model[Species] or Null.
AssayVolume
Default Calculation: Automatically determined based on Volume and TargetConcentration option values.
Pattern Description: Greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
ConcentratedBuffer
The concentrated buffer which should be diluted by the BufferDilutionFactor in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
BufferDilutionFactor
The dilution factor by which the concentrated buffer should be diluted in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: If ConcentratedBuffer is specified, automatically set to the ConcentratedBufferDilutionFactor of that sample; otherwise, set to Null.
BufferDiluent
The buffer used to dilute the aliquot sample such that ConcentratedBuffer is diluted by BufferDilutionFactor in the final solution. The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is specified; otherwise, resolves to Null.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
AssayBuffer
The buffer that should be added to any aliquots requiring dilution, where the volume of this buffer added is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is not specified; otherwise, resolves to Null.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
AliquotSampleStorageCondition
The non-default conditions under which any aliquot samples generated by this experiment should be stored after the protocol is completed.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
DestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
Pattern Description: Any well from A1 to H12 or list of one or more any well from A1 to H12 or any well from A1 to H12 entries or Null.
Programmatic Pattern: ((WellPositionP | {((Automatic | Null) | WellPositionP)..}) | Automatic) | Null
AliquotContainer
The desired type of container that should be used to prepare and house the aliquot samples, with indices indicating grouping of samples in the same plates, if desired. This option will resolve to be the length of the SamplesIn * NumberOfReplicates.
Default Calculation: Automatically set as the PreferredContainer for the AssayVolume of the sample. For plates, attempts to fill all wells of a single plate with the same model before aliquoting into the next.
Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null or {Index, Container} or list of one or more an object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null entries or list of one or more Automatic or Null or {Index, Container} entries.
Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null) | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | {((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null))..} | {({GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | (Automatic | Null))..}) | Automatic) | Null
AliquotPreparation
Default Calculation: Automatic resolution will occur based on manipulation volumes and container types.
ConsolidateAliquots
Protocol Options
Organizational Information
Template
A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.
Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.
Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null
Name
A object name which should be used to refer to the output object in lieu of an automatically generated ID number.
Post Experiment
MeasureWeight
Indicates if any solid samples that are modified in the course of the experiment should have their weights measured and updated after running the experiment. Please note that public samples are weighed regardless of the value of this option.
MeasureVolume
Indicates if any liquid samples that are modified in the course of the experiment should have their volumes measured and updated after running the experiment. Please note that public samples are volume measured regardless of the value of this option.
ImageSample
Example Calls
Basics
Reference Target
A gene (such as a housekeeping gene), whose expression should not be different between samples, can be amplified using optional primers and probes:
Active Well
Master Mix
For each sample, specify stock solution containing enzymes, buffer and nucleotides used to amplify DNA or RNA target sequences to be added to the reaction:
Reverse Transcription
A reverse transcription step to convert RNA target sequences to cDNA at the beginning of the thermocycling profile:
Gradient Annealing
A gradient annealing step to create a temperature gradient with minimum temperature at Row A and maximum temperature at Row H in the thermocycling profile to optimize binding of primers to template genes:
Sample Dilution
Generate a serial dilution curve to dilute the sample. Provide the volume of the sample, a dilution factor and the number of times the sample should be diluted. This should be used if you want the sample to be diluted by the same factor at each step of the serial dilution. The volume of the sample will be the final volume of the sample after the dilution is performed. The example call below will create a series of samples with dilution factors of (0.7, 0.49, 0.34, 0.24, 0.17, 0.12) and a volume of 60 Microliters.
Generate a serial dilution curve to dilute the sample. Provide the volume of the sample and a series of dilution factors the sample should be diluted by at each step. This should be used if you want the sample to be diluted by different factors at each step of the serial dilution. The volume of the sample will be the final volume of the sample after the dilution is performed. The example call below will create a series of samples with dilution factors of (0.7, 0.49, 0.34, 0.31, 0.28, 0.25) and a volume of 60 Microliters.
Generate a serial dilution curve to dilute the sample. Provide the volume of the sample that should be transferred, the volume of diluent the sample should be added to and the number of times the sample should be diluted. This should be used if you want give the volumes the sample will be diluted by with instead of dilution factors. The example call below will create a series of samples with dilution factors of (0.67, 0.44, 0.30, 0.20, 0.13, 0.09) and a volume of 50 Microliters.
Warnings and Errors
Messages (130)
ActiveWellOptionLengthMismatch (2)
ActiveWellOverConstrained (1)
DeprecatedModels (2)
DigitalPCRAmplitudeMultiplexing (2)
DigitalPCRDilutionSampleVolume (2)
When SampleDilution is True, volume of diluted samples to be created must be higher than SampleVolume*1.1 to ensure that sufficient dilute sample volume will be created:
When SampleDilution is True and NumberOfReplicates is specified, volume of diluted samples to be created must be higher than SampleVolume*NumberOfReplicates*1.1 to ensure that sufficient dilute sample volume will be created:
DigitalPCRDropletCartridgeLengthMismatch (1)
DigitalPCRDropletCartridgeObjectMismatch (1)
DigitalPCRDropletCartridgeRepeatedObjects (1)
DigitalPCRForwardPrimerConcentrationVolumeMismatch (2)
DigitalPCRForwardPrimerStockConcentration (3)
Forward primer oligomer concentration must be in units of Molar Concentration or Mass Concentration:
When forward primer oligomer concentration is in units of Mass Concentration, MolecularWeight is informed in the respective Model[Molecule,Oligomer] object:
Primer input object must have at least one oligomer in its composition:
DigitalPCRForwardPrimerStockConcentrationAccuracy (1)
DigitalPCRForwardPrimerStockConcentrationTooLow (1)
DigitalPCRForwardPrimerStorageConditionMismatch (2)
DigitalPCRInvalidPreparedPlate (3)
When using PreparedPlate, samples should be in Model[Container,Plate,DropletCartridge,"Bio-Rad GCR96 Digital PCR Cartridge"]:
When using PreparedPlate, primerPairs, probes, ReferencePrimerPairs and ReferenceProbes should be Null:
When using PreparedPlate with samples in a droplet cartridge, any wells without sample input must contain passive sample required for droplet generation in block of 16 wells in parallel:
DigitalPCRMasterMixConcentrationFactorNotInformed (1)
DigitalPCRMasterMixMismatch (2)
DigitalPCRMasterMixQuantityMismatch (1)
DigitalPCRMasterMixStorageConditionMismatch (1)
DigitalPCRMultiplexedTargetQuantity (1)
DigitalPCRMultiplexingNotAvailable (2)
DigitalPCRNonLiquidSamples (3)
DigitalPCROverConstrainedRowOptions (2)
DigitalPCRPremixedPrimerProbe (1)
DigitalPCRPrimerGradientAnnealingMismatch (1)
DigitalPCRPrimerGradientAnnealingRowTemperatureMismatch (1)
DigitalPCRPrimerProbeMismatchedOptionLengths (1)
DigitalPCRProbeConcentrationVolumeMismatch (2)
DigitalPCRProbeStockConcentration (2)
DigitalPCRProbeStockConcentrationAccuracy (1)
DigitalPCRProbeStockConcentrationTooLow (1)
DigitalPCRProbeStorageConditionMismatch (1)
DigitalPCRProbeWavelengthsIncompatible (1)
DigitalPCRProbeWavelengthsLengthMismatch (1)
DigitalPCRProbeWavelengthsNull (2)
DigitalPCRReferenceForwardPrimerConcentrationVolumeMismatch (2)
DigitalPCRReferenceForwardPrimerStockConcentration (2)
DigitalPCRReferenceForwardPrimerStockConcentrationAccuracy (1)
DigitalPCRReferenceForwardPrimerStockConcentrationTooLow (1)
DigitalPCRReferenceForwardPrimerStorageConditionMismatch (2)
DigitalPCRReferencePremixedPrimerProbe (1)
DigitalPCRReferencePrimerProbeMismatchedOptionLengths (1)
DigitalPCRReferenceProbeConcentrationVolumeMismatch (2)
DigitalPCRReferenceProbeStockConcentration (2)
DigitalPCRReferenceProbeStockConcentrationAccuracy (1)
DigitalPCRReferenceProbeStockConcentrationTooLow (1)
DigitalPCRReferenceProbeStorageConditionMismatch (1)
DigitalPCRReferenceProbeWavelengthsIncompatible (1)
DigitalPCRReferenceProbeWavelengthsLengthMismatch (1)
DigitalPCRReferenceProbeWavelengthsNull (1)
DigitalPCRReferenceReversePrimerConcentrationVolumeMismatch (2)
DigitalPCRReferenceReversePrimerStockConcentration (2)
DigitalPCRReferenceReversePrimerStockConcentrationAccuracy (1)
DigitalPCRReferenceReversePrimerStockConcentrationTooLow (1)
DigitalPCRReferenceReversePrimerStorageConditionMismatch (3)
ReferenceReversePrimerStorageCondition cannot have conflicts with the reverse primer input in ReferencePrimerPair:
When using any TargetAssay, ReferenceReversePrimerCondition cannot have conflicts with ReferenceProbeStorageCondition:
When using any PrimerSet, ReferenceReversePrimerCondition cannot have conflicts with ReferenceForwardPrimerStorageCondition:
DigitalPCRReversePrimerConcentrationVolumeMismatch (2)
DigitalPCRReversePrimerStockConcentration (2)
DigitalPCRReversePrimerStockConcentrationAccuracy (1)
DigitalPCRReversePrimerStockConcentrationTooLow (1)
DigitalPCRReversePrimerStorageConditionMismatch (3)
DigitalPCRSampleDilutionMismatch (7)
When SampleDilution is True, SerialDilutionCurve cannot be Null:
When SampleDilution is True, DilutionNumberOfMixes cannot be Null:
When SampleDilution is True, DilutionMixVolume cannot be Null:
When SampleDilution is True, DilutionMixRate cannot be Null:
When PreparedPlate is True, SampleDilution cannot be True:
When SampleDilution is False, SerialDilutionCurve cannot be specified:
When SampleDilution is False, DilutionMixVolume cannot be specified:
DigitalPCRSingleFluorophorePerProbe (2)
DigitalPCRSingleReferenceFluorophorePerProbe (2)
DigitalPCRTotalVolume (3)
Sum of volumes of all stock solutions matches the specified ReactionVolume:
Total volume with all components matches ReactionVolume:
When Model[Sample,"Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes, Buffer"] (concentration factor = 4) is used as MasterMix, total volume calculation adjusts for the use of Model[Sample,StockSolution,"Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes, 2x Master Mix"] (concentration factor = 2) when calculating the total volume:
DiscardedSamples (2)
InvalidActiveWells (2)
InvalidInputSampleContainer (2)
InvalidPrimerProbeInputs (1)
ObjectDoesNotExist (8)
Throw a message if we have a sample that does not exist (name form):
Throw a message if we have a container that does not exist (name form):
Throw a message if we have a sample that does not exist (ID form):
Throw a message if we have a container that does not exist (ID form):
Do NOT throw a message if we have a simulated sample but a simulation is specified that indicates that it is simulated:
Do NOT throw a message if we have a simulated container but a simulation is specified that indicates that it is simulated:
Do NOT throw a message if we have a simulated sample/container as primer but a simulation is specified that indicates that it is simulated:
Do NOT throw a message if we have a simulated sample/container as probe but a simulation is specified that indicates that it is simulated:
PassiveWellsAssignment (1)
ThermocyclingOptionsMismatchInPlate (2)
Possible Issues
Insufficient number of droplets detected
Statistical analysis of droplet populations requires signal readings from at least 10,000 droplets. Insufficient reaction volume, use of non-standard master mixes or errors in assay plate loading may result in issues with droplet generation.
One-step reverse transcription
RNA templates are highly unstable and may result in poor quality of target amplification. Rapid processing of samples after extraction is critical for achieving successful results.
Poor separation of droplet populations for amplitude multiplexed targets
Multiplexing of several target and reference genes in a single channel may result in insufficient separation of droplet populations. Optimization of primer and probe concentrations to achieve adequate signal separation for different populations, optimization of primer sequences to improve target amplification, or optimization of thermocycling parameters, may be required to improve data quality.
Last modified on Fri 5 Sep 2025 09:00:35