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Trusight HLA

Best practices

 

Ensuring Consistency

  • To ensure consistency across samples, use a multi-channel pipette where possible. Calibrate pipettes periodically.
  • To avoid unnecessary freeze-thaw cycles when performing experiments of fewer than 96 samples, Illumina recommends that you aliquot smaller volumes of reagents normally stored frozen after they are thawed for the first time.

 

Avoid Cross-Contamination

  • Always use fresh pipette tips between samples and between dispensing index primers.
  • Mix samples with a multi-channel pipette and centrifuge the plate when indicated. Do not vortex the plates.
  • Pipette carefully to avoid spillage.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.
  • When adding samples to a 96-well plate, change tips between each sample.
  • When adding HLA primers to a 96-well plate, change tips between each row.
  • When adding i7 index adapters to columns of a 96-well plate, change tips between each column.
  • When adding i5 index adapters to rows of a 96-well plate, change tips between each row.
  • When finished adding i7 index adapters, replace each cap with a new orange cap.
  • When finished adding i5 index adapters, replace each cap with a new white cap.
  • After index adapters have been added to the 96-well plate, remove unused index adapter tubes from the working area.

 

Sealing the Plate

  • Always seal the 96-well plate before the following steps in the protocol:
    • Shaking steps
    • Centrifuge Steps
    • Thermal Cycling Steps
  • Apply the adhesive seal to cover the plate and seal with a rubber roller.
  • Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semi-skirted PCR plates.
  • Foil seals are effective are effective at -70°C to 105°C, and suitable for skirted or semiskirted PCR plates.
  • Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

 

About Reagents

  • RSB (Resuspension Buffer)—When adding RSB to a 96-well plate that contains SPB, add the RSB when the plate is on the magnetic stand. The dry pellet can carry static electricity, which is discharged with the addition of RSB.
  • LNA1 (Library Normalization Additive)—LNA1 can form visible precipitates or crystals. Before use, bring to room temperature, vortex vigorously, and then visually inspect in front of a light to make sure that all precipitate has dissolved.
  • LNB1 (Library Normalization Beads)—Before use, bring to room temperature, vortex vigorously at least 1 minute, and then invert the tube to mix. Make sure that no pellet is present at the bottom of the tube when inverted. Mix only the amounts of LNA1 and LNB1 required for the current experiment. Store remaining LNA1 and LNB1 separately at their respective temperatures.
  • SPB (Sample Purification Beads)—Before use, bring to room temperature and vortex.

 

Plate Transfers

  • When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.
  • When multiple plates are used in a step, such as a plate 1 and a plate 2, transfer volumes from the existing plate 1 to the new plate 1. Transfer volumes from the existing plate 2 to the new plate 2.

Handling Magnetic Beads

  • Prior to use, allow the beads to come to room temperature.
  • Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogeneous.
  • Take care to minimize bead loss which can impact final yields.
  • Change the tips for each sample.
  • Let the mixed samples incubate at room temperature for the full duration specified in the protocol to ensure maximum recovery.
  • When aspirating the cleared solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and to not disturb the separated magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips.
  • To prevent the carryover of beads after elution, approximately 2.5 μl of supernatant are left when the eluates are removed from the bead pellet.
  • Always prepare fresh 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh 80% ethanol should be prepared fresh for optimal results.
  • Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants.
  • Keep the reaction plate on the magnetic stand and let it air-dry for 2 minutes at room temperature to prevent potential bead loss due to electrostatic forces. Allow for the complete evaporation of residual ethanol, as the presence of ethanol impacts the performance of the subsequent reactions. 
  • Resuspend the dried pellets using a single channel or multi-channel pipette.
  • When removing and discarding supernatant from the wells, use a single channel or multi-channel pipette and take care not to disturb the beads.
  • To maximize DNA recovery during elution, incubate the DNA/bead mix for two minutes at room temperature before placing the samples onto the magnet.