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5_Cleanup.md

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Protocol 5: Pool cleanup

Theory

Before sequencing, it is crucial to remove any free adapter and/or primer dimers which will cause a loss of reads during sequencing and/or resulting in enhanced barcode hoping. For more on the idea of barcode hoping, see here. This protocol can be accomplished in one of two ways: Ampure XP bead cleanup (Protocol A) or gel extraction (Protocol B). Note: an automated size selection method such as a pippin prep can be used as well if available.

Materials

Protocol A

  • MinElute Gel Extraction Kit (Qiagen 28604)
  • TBE or TAE (BioRad 1610773)
  • Agarose for Molecular Biology (BioRad 1613100)
  • Quick-Load 100bp DNA Ladder (NEB N0467S)

Protocol B

  • Ampure XP beads (Beckman A63881)
  • Freshly made 80% ethanol
  • Nuclease Free Water
  • Magnetic capture rack

Protocol A: Gel purification

  • Prepare 50mL of 1% agarose (0.5g) in the microwave using running buffer.
  • Add 5 µL of SYBR Safe stain to the gel and mix before pouring into freshly cleaned (with RNase Away or similar) gel mold. Use the smallest lane width possible.
  • Mix 24ul library with loading buffer (according to concentration of buffer).
  • Load gel along with 5µL of ladder leaving a space between wells.
  • Run for ~45 minutes at 80V.
  • Using blue light tray, excise the band at 435bp into a empty preweighed tube
  • Weigh mass of gel and add 4 volumes Gel Dissolving Buffer (Biolab Monarch Kit).
  • Incubate for 5-10min @ 50˚C and/or vortexing periodically until gel has dissolved.
  • Insert column into collection tube and load sample onto the column.
  • Spin for 1 minute at 10,000g, then discard flow-through.
  • Insert column back to the collection tube. Add 200uL DNA Wash Buffer and spin for 1 minute.
  • Discard flow through and repeat the previous step.
  • Transfer column into a clean 1.5mL microcentrifuge tube and SPIN for 1 minute to remove the excess ethanol.
  • Add 12uL of DNA elution Buffer to the center of the matrix without touching the mesh. Spin for 1 minute to elute DNA.
  • Quantify by Nanodrop and Qubit.

Protocol B: Magnetic Capture

  • Mix 100ul of pooled library with 80µL ampure XP beads (0.8x bead volume has been picked to remove adapter dimers and free adapter)
  • Incubate 10 min at room temperature
  • Capture for ~ 3 min
  • Discard supernatant
  • Add 200 ul fresh 80% ethanol, briefly mix and recapture
  • Discard ethanol
  • Add 200 ul fresh 80% ethanol, briefly mix and recapture
  • Discard ethanol
  • briefly centrigue, recapture, and use pipette to remove any ethanol at bottom of tube
  • Airdry beads for ~ 5 min at room temperature until no visible ethanol remains
  • Add 35ul nuclease free water and mix well
  • Capture beads
  • Transfer supernatant to fresh tube and quantify using Nanodrop and/or Qubit