Transcriptic

Thermocycling

Instruction and Parameters

A thermocycle step can either be at a constant temperature or with a gradient applied row-wise down the plate. Constant temperatures can range from 0°C - 100°C, and gradient temperatures can range from 30°C - 100°C. Additionally, the difference between the top and bottom temperatures in a gradient step must range from 1°C - 24°C, with the top parameter being the greater temperature. Temperatures and durations can be changed in increments of 0.1°C and 1 second, respectively.

{
  "op": "thermocycle",
  "object": container,
  "volume": volume,
  "groups": [{
    "cycles": integer,
    "steps": [{
      "duration": duration,
      "temperature": temperature,
      "read": boolean // optional (default false)
    },{
      "duration": duration,
      "gradient": {
        "top": temperature,
        "bottom": temperature
      },
      "read": boolean // optional (default false)
    }]
  }],

  // optionally
  "dyes": {
    <dye_name>: [well]
  },
  "dataref": string,
  "melting": {
    "start": temperature,
    "end": temperature,
    "increment": temperature,
    "rate": duration
  }
Plate type
Maximum volume

96-pcr

50uL

384-pcr

30uL

The dyes object is a map from a selected fluorophore to the list of wells that contain it. Both the dyes and dataref objects are not specified in a regular thermocycle instruction, but must be specified if any real-time measurements are to be specified. Any of the steps of a thermocycle group may enable an optional boolean read parameter to signal a qPCR reading after its corresponding step. The default value for read is false.

For real-time measurements, the available dyes are:

Channel 1
Excitation range: 450nm - 490nm
Emission range: 510nm - 530nm
Detectable dyes: FAM,SYBR
Channel 2
Excitation range: 515nm - 535nm
Emission range: 560nm - 580nm
Detectable dyes: VIC,HEX,TET,CALGOLD540
Channel 3
Excitation range: 560nm - 590nm
Emission range: 675nm - 690nm
Detectable dyes: ROX,TXR,QUASAR670
Channel 4
Excitation range: 620nm - 650nm
Emission range: 675nm - 690nm
Detectable dyes: CY5,QUASAR670
Channel 5
96-well plates only
Excitation range: 672nm - 684nm
Emission range: 705nm - 730nm
Detectable dyes:QUASAR705

An optional melting object may be included at the end of the thermocycle instruction. It will take its dye configuration from the top-level dyes object. Its temperature increment parameter may take values from 0.1°C - 9.9°C. Note that a melt has an implicit 30 second hold at its beginning.

The volume parameter denotes the sample volume, which is used to estimate the sample temperature. This helps to maintain consistency between the intended and actual behavior of thermocycle instructions. The value of volume must be between 0uL - 50uL for 96 well plates and 0uL - 30uL for 384 well plates, and it will be rounded to the nearest microliter.

Plates must be sealed using the seal instruction before any thermocycler instructions can be executed. Submitting a protocol that thermocycles an unsealed plate will yield an error message.

Device

All thermocycle instructions will be executed on a Bio-Rad CFX96 or CFX384 system depending on the plate type used. The per minute costs are tiered, depending on your use of the quantitation system.

Standard Operation

The average ramp rate of the CFX thermocyclers is 3.3°C with a maximum of 5°C. Gradients can be run from 30°C to 100°C with a total temperature span between 1°C and 24°C. The temperature accuracy has been determined to be ±0.2°C of the target temperature with a well-to-well uniformity of ±0.4°C within 10 seconds of arriving at the target temperature.

Optics

The optics use 6 filtered LEDs and photodiodes. Using LEDs provides some major advantages over lamp-based systems. Lamp-based systems are passive and as such the light path lengths is different for every well. A well with a shorter light path will have a higher fluorescence reading compared to a well with a longer path. To compensate for this effect the data has to be normalized by a reference dye. The LED based system does not require that, as the LEDs are positioned directly over each well - resulting in equal length light path. More details on the devices can be found on the Bio-Rad website.