Best Practices

General Advice on Sample Handling

  • To minimize sample loss throughout the Nextera Mate Pair Sample Preparation wehighly recommend using Axygen Maxymum Recovery 1.7 ml tubes (Cat # MCT-175-LC),for their low retention and heat resistance properties.
  • When adding and mixing reagents or bead wash solutions, try to avoid mixingreactions by pipetting up and down, as small amounts of sample may remain in thetip and be lost. Instead add the reagents or wash buffers and mix the solution byflicking the sample tube. Then briefly spin the sample in a mini-centrifuge to collectsample to the bottom of tube.
  • When handling DNA samples with fragment sizes above 2 kb (all steps prior to ShearCircularized DNA), do not vortex samples to mix, as this may further shear the DNA.

Handling AMPure Magnetic Beads

Follow appropriate handling methods when working with AMPure XP Beads and theNextera Mate Pair Sample Preparation Protocol:

NOTE: AMPure purification procedures in this user guide have only been validatedusing the Axygen Maxymum Recovery 1.7 ml microcentrifuge tubes and themagnetic stand specified in the Consumables and Equipment list. Comparableperformance is not guaranteed when using other microcentrifuge tubes or tubeformats, or other magnets.

  • Prior to use, allow the beads to come to room temperature.
  • Do not reuse beads. Always add fresh beads when performing these procedures.
  • Pipette carefully to avoid spillage.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.

Handling Magnetic Beads

  • The M280 Dynabeads beads do not need to come to room temperature before use.
  • Do not reuse beads. Always add fresh beads when performing these procedures.
  • Immediately prior to use, mix the beads well by shaking, until they are well dispersed.The color of the liquid should appear homogeneous.
  • Once the DNA has been bound to the Streptavidin beads, it remains bound to thebeads until it is amplified off the beads in the PCR amplification reaction.
  • The following steps are all carried out on-bead: End Repair, A-Tailing, AdapterLigation, and PCR Amplification.
  • To avoid unnecessary sample loss due to pipette tip carryover, do not resuspend thebeads using a pipette, instead resuspend them by flicking and then spin in a minicentrifugeto collect the sample to the bottom of the tube.
  • When washing the beads during the on-bead reaction steps, please note that theResuspension Buffer is used as a wash buffer and does not elute the DNA from thebeads.
  • When washing the beads after the on-bead reactions, resuspend the beads in the washbuffer by flicking tube multiple times, and then spin in a mini-centrifuge. Bycentrifuging the bead/wash solution for a longer period of time (up to 10 seconds), thebeads can be collected to the bottom of the tube, thus aiding the subsequent collectionof the beads to the magnet.
  • Before adding the on-bead reaction mixtures, remove as much of the finalResuspension Buffer wash solution as possible with a 200 μl pipette tip, spin the tubeagain briefly, and place back on the magnet. Then remove any remainingResuspension Buffer with a 10 μl pipette.
  • After adding the on-bead reaction mixtures, resuspend the beads in the reactionmixture by flicking the tube multiple times, and then spin very briefly in a minicentrifuge.Centrifuge the reactions only long enough to collect the solution to thebottom of the tube, but not so long as to begin to pellet the beads (approximately 1-2seconds at low speed).
  • During the on-bead reaction incubations the beads will settle to the bottom of the tube,this is normal and will not affect library preparation.

Handling 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 inorder to avoid excessive freeze-thaw cycles. However, if you aliquot, you might nothave enough reagents for the full number of reactions over multiple uses.
  • Add reagents in the order indicated.
  • Due to their viscosity, take extra care while adding the following reagents; Mate PairTagment Enzyme, Strand Displacement Polymerase, Circularization Ligase, End RepairMix, A-Tailing Mix, and Ligation Mix.