Best Practices

Best Practices

Sealing a Plate

  • Always seal the 96-well plate before the following steps in the protocol:
    • Shaking
    • Vortexing
    • Centrifuge
  • 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 semiskirted PCR plates.

Covaris Tubes and Instruments

  • Make sure that pipettes are calibrated before beginning. Uncalibrated pipettes can lead to variations in insert size, and reagents not dispensing properly, resulting in run failure.
  • To ensure reproducible DNA shearing, centrifuge samples before fragmenting.
  • Load DNA samples into the Covaris tube slowly to avoid creating air bubbles. However, air bubbles might not be preventable.
  • Use the wells of a plate or another device to hold the Covaris tubes upright.
  • Remove the Intensifier from the E220 instrument before fragmenting with a microTUBE‑15.
  • If using a Covaris instrument other than those specified, contact Covaris.

Handling the Library Card

  • To avoid instrument damage, do not place the library card guide on the library card during library card verification or a run.
  • Use the library card latch release to load and remove the library card from the library card stage.
    • Do not snap the library card into place.
    • Oil and reagents in a used library card can splash out of the card and onto the instrument.
  • Hold the used library card level when removing it from the instrument to avoid spilling its contents.

Library Card Loading Guidelines

  • Load the library card while it is on the library card stage to avoid spilling or disturbing the loaded contents.
  • The NeoPrep Control Software guides you through the steps to set up a run and load the library card. Use the loading procedures in this guide as a reference.
  • Do not open the compartment door during library card verification or a run.
  • Change your gloves after loading the oil.
  • Transfer contents from the reagent plate to the corresponding wells on the library card.
  • Reference the corresponding colors and well labels on the reagent plate and library card guides.
  • Use the pipettes and tips specified in the TruSeq Nano DNA Library Prep for NeoPrep Reference Guide. Other pipettes and tips are not supported and can result in reagents not dispensing properly and run failure.
  • Use a multichannel pipette to load reagents, samples, and adapters.
  • To avoid instrument damage, make sure that the library card guide is removed from the library card before starting the run.

Library Card Loading Techniques

  • Use proper library card loading techniques and specified loading angles.
  • Pipette to the first stop to avoid creating bubbles.
  • Insert pipette tips perpendicular to the well.
  • Insert the pipette tips to the bottom of the well while dispensing. Do not lift the tips until the reagents are dispensed completely.
  • Dispense at an angle by pointing pipette tips under the well label and dotted well outline on the library card guide.
  • The pipette loading angle depends on the item being dispensed. The angle is specified in each step of the NeoPrep Control Software loading guide and is depicted in the TruSeq Nano DNA Library Prep for NeoPrep Reference Guide.
  • An icon represents the loading angle and the volume is specified on the NeoPrep Control Software loading guide.
  • Increase the pipette angle if liquid is not dispensing from the pipette tips.

Handling Samples

  • Always track the location of each sample.
  • Change tips between each sample to avoid cross-contamination.
  • Do not centrifuge samples before loading.
  • Some space might be present in the pipette tips during transfer from the sample plate to the library card.

Collecting Libraries

  • Unload the library card while it is on the library card stage.
  • The NeoPrep Control Software guides you through the steps to unload the library card. Use the TruSeq Nano DNA Library Prep for NeoPrep Reference Guide as a reference.
  • Do not use a 20 µl pipette. It does not fit properly into the library card well.
  • Insert pipette tips perpendicularly and touch the tips to the bottom of the collection wells.
  • Hold down the library card with one hand while removing the tips from the collection ports to prevent any movement of the card.
  • An icon represents the required pipette angle, and the volume is specified on the NeoPrep Control Software unloading guide.
  • Inspect each pipette tip to make sure that a blue library droplet is present in the tips indicated by the NeoPrep Control Software.
  • If a blue library droplet is not visible in each expected pipette tip, do the following:
    • Transfer the extracted liquid to the corresponding plate well containing RSB.
    • Do not dispense the liquid back into the library card, which can introduce air gaps and interfere with library extraction.
    • Use a single-channel pipette to repeat the transfer 1 time for the wells that did not contain the blue droplet. Do not attempt the transfer more than 2 times.
  • Vigorously pipette up and down in RSB to dislodge the blue library droplet from the pipette tip.

Handling Library Separation Tube Strips

  • Label the tubes to support tracking sample location.
  • Use the wells of a plate or another device to hold the library separation tube strips upright.
  • Do not centrifuge library separation tube strips.