Best Practices

Best Practices

Consistency

  • To make sure there is consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.
  • To minimize sample loss from manual resuspension, use the recommended plates and plate shaker to mix samples.

Prevent PCR Product Contamination

  • Make sure that the lab is set up appropriately to reduce the risk of PCR product contamination. 

Avoid Cross-Contamination

  • Open only one adapter at a time.
  • Using aerosol-resistant pipette tips reduces the risk of nucleic acid carry-over and sample-to-sample cross-contamination. If aerosol-resistant tips are not available, ensure careful pipetting to avoid spillage and contamination.
  • Always use fresh pipette tips between samples and between dispensing index primers, unless specified otherwise.
  • Mix samples using the methods specified in the protocol.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.

Handling RNA

RNA is highly susceptible to degradation by RNase enzymes. RNase enzymes are present in cells and tissues, and carried on hands, labware, and dust. They are stable and difficult to inactivate. Follow these best laboratory practices while preparing and handling RNA samples:

  • When harvesting total RNA, use a method that quickly disrupts tissue and isolates and stabilizes RNA.
  • Always wear gloves and use sterile technique.
  • Reserve a set of pipettes for RNA work. Use sterile RNase-free filter pipette tips to prevent cross-contamination.
  • Use disposable plasticware that is certified to be RNase-free. Nonsticky sterile RNase-free microcentrifuge tubes are recommended. Designate a set of these tubes for this protocol. Do not use these tubes for other lab work.
  • Prepare all reagents from RNase-free components, including ultrapure water.
  • Store RNA samples by freezing. Avoid extended pauses in the protocol until the RNA is in the form of double-stranded DNA (dsDNA).
  • Use a RNAse/DNAse decontamination solution to decontaminate work surfaces and equipment before starting this protocol.

Temperature Considerations

  • Keep libraries at temperatures ≤ 37°C, except where specifically noted in a protocol.
  • Place reagents on ice after thawing at room temperature.
  • When processing more than 48 samples manually, Illumina recommends processing the plate on a bed of ice whenever possible, especially during enzymatic steps (when using the A-Tailing Mix and Ligation Mix). A large number of samples processed at room temperature may result in uneven catalytic activity, which can lead to reduced quality of the end product.
  • Avoid elevated temperatures, particularly in the steps preceding the adapter ligation.
  • Temperature is less of an issue after the adapters have been ligated onto the ends of the ds cDNA.

Handling Liquids

Good liquid handling measures are essential, particularly when quantifying libraries or diluting concentrated libraries for making clusters.

  • Small differences in volumes (±0.5 µl) can sometimes give rise to very large differences in cluster numbers (~100,000).
  • Small volume pipetting can be a source of potential error in protocols that require generation of standard curves, such as PicoGreen assays or qPCR, or those that require small but precise volumes, such as the Agilent Bioanalyzer.
  • If small volumes are unavoidable, then due diligence should be taken to ensure that pipettes are correctly calibrated.
  • Make sure that pipettes are not used at the volume extremes of their performance specifications.
  • When adapting this protocol for automation or robots, process >16 samples to minimize reagent loss due to dead volume.

Handling Master Mix Reagents

  • Minimize freeze-thaw cycles. If you do not intend to consume the reagents in one use, dispense the reagent into aliquots after the initial thaw and refreeze the aliquots in order to avoid excessive freeze-thaw cycles. However, if you aliquot, you may not have enough reagents for the full number of reactions over multiple uses.
  • Add reagents in the order indicated and avoid making master mixes containing the in-line controls.
  • Take care while adding ATL (A-Tailing Mix) and LIG (Ligation Mix) due to the viscosity of the reagents.
  • Centrifuge the master mix reagents to 600 × g for 5 seconds before use.
  • The kit contains First Strand Synthesis Mix Act D (FSA) which contains Actinomycin D, a toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the product safety data sheet (SDS) for detailed environmental, health, and safety information.

Handling Magnetic Beads

Follow appropriate handling methods when working with AMPure XP and RNAClean XP Beads:

  • Prior to use, allow the beads to come to room temperature.
  • Do not reuse beads. Always add fresh beads when performing the procedures.
  • Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogeneous.
  • After adding the beads to the reaction, seal the plate and shake the plate on a microplate shaker at 1800 rpm for 2 minutes. Repeat, if necessary, until the color of the mixture appears homogeneous after mixing.
  • 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, as required in the protocol. Ethanol can absorb water from the air impacting your results.
  • Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants. Remaining ethanol can be removed with a 20 μl pipette.
  • Keep the reaction plate on the magnetic stand and let it air-dry at room temperature to prevent potential bead loss due to electrostatic forces.
  • Allow at least 5 minutes of drying time on the magnetic stand at room temperature for complete evaporation. Residual ethanol can impact the performance of subsequent reactions.
  • Do not exceed the recommended drying time as overdrying samples can negatively impact sample recovery and over drying the beads can impact final yields .
  • Resuspend the dried pellets by shaking.
  • When removing and discarding supernatant from the wells, use a single channel or multichannel pipette and take care not to disturb the beads.
  • To maximize sample recovery during elution, incubate the DNA/bead mix for 2 minutes at room temperature before placing the samples onto the magnet.

Equipment

  • Review the programming instructions for your thermal cycler user guide to ensure that it is programmed appropriately using the heated lid function.
  • Calibrate the microplate shaker with a stroboscope.