Transcriptic

Magnetic Transfer

Facilitates a number of operations that involve magnetic bead separations.

Beta

This instruction is currently in beta development. Physical movement of the magnetic head is under full control of autoprotocol, however, magnetic bead purification applications have not been validated on the system. Development of your own magnetic bead purifications should proceed with care and caution and may take some time to optimize.

Magnetic transfer at Transcriptic uses magnetic pins covered in disposable sheaths to move beads from plate to plate, which can contain different solutions, to allow for sample collection, washing, and elution. Magnetic transfer differs from traditional magnetic bead purification where beads remain in a single plate and solutions are transferred in and out of that plate.

Instruction and Parameters

The magnetic_transfer operation accepts an array of groups of one or more sub-operations. These are collect, mix, release, dry or incubate.

magnetic_head accepts one of two options set depending on the plate being used with the operation. The options are "96-deep" or "96-pcr".

96-deep is compatible with containers of type 96-v-kf, 96-deep-kf, 96-deep.

96-pcr, head is compatible with containers of type 96-pcr, 96-v-kf, 96-flat, 96-flat-uv.

{
  "op": "magnetic_transfer",
  "groups": [
    /* sub-operations in the same group use the same protector tip */
    [
      {
        /* tips are always magnetized during collect */
        "collect": {
          "object": container,
          "cycles": integer,
          /* time spent paused at "bottom_position" */
          "pause_duration": time,
          /* position relative to well height where tips pause,
             optional, default: 0.0 */
          "bottom_position": float,
          /* optional, default: null */
          "temperature": temperature  
        }
      },
      {
        "mix": {
          "object": container,
          "duration": time,
          "frequency": frequency,
          /* optional, default: false */
          "magnetize": bool,
          /* optional, default: null */
          "temperature": temperature,
          /* position relative to well height where oscillation is centered,
             optional, default: 0.5 */
          "center": float,
          /* distance relative to well height to oscillate around "center", 
             optional, default: 0.5 */
          "amplitude": float
        }
      },
      {
        /* tips are never magnetized during release */
        "release": {
          "object": container,
          "duration": time,
          "frequency": frequency,
          /* optional, default: null */
          "temperature": temperature,
          /* position relative to well height where oscillation is centered,
             optional, default: 0.5 */
          "center": float,
          /* distance relative to well height to oscillate around "center", 
             optional, default: 0.5 */
          "amplitude": float
        }
      },
      {
        /* tips are always magnetized during dry,
           tips are held above the "object" for the indicated "duration" */
        "dry": {
          "object": container,
          "duration": time
        }
      },
      {
        "incubate": {
          "object": container,
          "duration": time,
          /* optional, default: false */
          "magnetize": bool,
          /* position relative to well height that tips are held, 
             optional, default: 1.5 */
          "tip_position": float,
          /* optional, default: null */
          "temperature": temperature
        }
      }, ...
    ], ...
  ],
  "magnetic_head": "96-deep" | "96-pcr"
}

Sub-Operations

All sub-operations within the same group will use the the same tip sheath and sheaths are changed between different groups of sub-operations.

collect - used to collect beads from a whole container object. Protection tips are magnetized and raised and lowered repeatedly pausing at the bottom_position for pause_duration time. The lowering, pausing, and raising is performed a number of cycles times. During this instruction, the object can be optionally heated to temperature.

mix - used to oscillate the tips vertically in an object. The oscillation will last for duration and move with a frequency and amplitude around center. During this instruction, the object can be optionally heated to temperature and the tips can be magnetized.

release - used to release beads into an object. Non-magnetized tips are oscillated vertically. The oscillation will last for duration and move with a frequency and amplitude around center. During this instruction, the object can be optionally heated to temperature.

dry - used to dry beads with tips above and outside an object for a set duration.

incubate - used to incubate an object for a set duration with the tips moved to tip_position. During this instruction, the object can be optionally heated to temperature and the tips can be magnetized.

Sub-Operation Parameters

The bottom_position, tip_position, center, and amplitude parameters are expressed as floats of relative well height. Setting one of these parameters to 0 represents positioning from the well bottom or height of 0, 1 represents positioning at the well top or the height of a well, and 1.5 represents positioning at 1.5 times the well height from the well bottom (or half well height above the well top).

frequency dictates the frequency with which the probes are inserted and retracted from the plate wells and is specified as a "float:hertz" unit. The lower bound of valid frequencies is 0, the upper bound varies on a number of parameters as follows:

max_frequency = 250[mm/s]/(2*amplitude*wellDepth)

Well Depths:
96-deep-kf = 42.3 mm
96-deep = 42.03 mm
96-v-kf = 12.8 mm
96-flat = 10.67 mm
96-pcr = 14.6 mm

The following table gives some basic recommendations for frequency and matching speeds on the Kingfisher Flex using an amplitude of 0.5.

Plate Type
Slow hertz
Medium hertz
Fast hertz

96-deep-kf

0.15

1.5

2.5

96-deep

0.15

1.5

2.5

96-v-kf

0.5

4.5

9.5

96-flat

0.5

5.5

11.5

96-pcr

0.4

4

8.5

If temperature is null or unset, no heat will be applied and the container will not be set onto the heating plate. Valid temperatures fall in the range of 30:celsius to 115:celsius.

Transcriptic currently supports the use of up to 8 containers and tip protectors per instruction. Specifically, the number of unique containers plus the number of groups in a single instruction must be less than or equal to 8.

Development Considerations

The "96-pcr" magnetic head is weaker than the "96-deep" magnetic head. Depending on liquid types and magnetic beads used, the strength of the "96-pcr" magnetic head may have difficulty attracting beads with enough strength to break surface tension for high surface tension liquids such as water. In this circumstance, the manufacturer has recommended the addition of a low concentration of detergent (e.g. 0.02% Tween) in order to assist in breaking surface tension. If problems with retrieval occur and 0.02% Tween does not interfere with your biology, please consider adding in detergent as a troubleshooting step. Increasing the number of collection steps and the length of collection time may also help with collecting the beads.

The "96-pcr" magnetic head does not completely form fit to the PCR wells which can result in inefficient mixing. Mixing speed can play a significant role in sample retrieval and medium speed has been recommended for many applications. Some applications may require beads to be mixed with the liquid handling instructions (single or multichannel) before use on the Kingfisher.

Magnetic collection works best with beads greater than 1μm in size. Beads smaller than that size may require more optimization.

Please be aware of the volume of reagents you are using with magnetic transfer instructions. Using too high a volume with a full height mix, could result in spillage, contamination, and/or loss of sample.

Containers

Container compatibility

For a full list of containers and compatibility see Containers

While a KingFisher Flex accepts a number of container types there are three containers specifically designed for high recoverability with the KingFisher: 96-deep-kf, 96-v-kf, and 96-pcr. For the properties of these plates see Containers.

For any collect sub-operations it is recommended to use a KingFisher specific container. In this situation it is advisable to stamp the entire source container to a KingFisher specific container.

For any of the sub-operations when temperature is being set it is recommended to use a KingFisher specific container.

Device

Magnetic bead handling is undertaken by a Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor. Samples are processed 96-wells at a time in a plate based format.

Please see the Kingfisher Site for more information.