Arichard:Protocols/MDA amplicon tagmentation and amplification

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MDA amplicon tagmentation and amplification[edit]

  • All volumes exact. Adjust for error.
  • High molecular weight buffer and qPCR tubes can be UV treated during ethanol precipitation if proceeding directly from extraction and processing.
  • A 1:1 bead:sample volume will capture DNA >= 200 bp, 1:1.8 will capture >= 100 bp.

Prep[edit]

  • UV treat 2 ul high-molecular weight (HMW) buffer per sample in a 0.2 ml tube, along with white qPCR tube strips and caps, 1 tube per sample.
  • UV treat 99 ul H2O for protease dilution.
  • UV treat 31 ul H2O per sample in a 1.5 ml tube for PCR master mix. This can be prepared during the Exo- reaction.

Protocol[edit]

Tagmentation[edit]

  1. Add 2 ul HMW buffer to each white qPCR tube.
  2. Transfer samples to white qPCR tubes.
  3. Add 1 ul transpose (1:50 dilution) to each sample. Incubate 5 minutes at 55 degC.
  4. Dilute protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC.
  5. Make the master mix for Exo- treatment: 1 ul Exo- and 1 ul dNTP per sample.
  6. Add 2 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC.

Amplification with primers and adapters[edit]

  1. Finish primer PCR master mix (per sample):
    1. 15 ul 2X KAPA2G Robust Hot Start.
    2. 0.6 ul Orange primer.
    3. 0.6 ul Blue primer.
    4. 0.24 ul 50X SYBR Green.
    5. 0.56 ul H2O.
  2. Add 17 ul primer PCR master mix to each sample.
  3. Place tubes in real time thermocycler (Login: Eppendorf, Password: e).
  4. Use Background: EP_WWStriptubes, Wavelength: 520 SYBR, Probe: SYBR, 100 ul reaction volume.
  5. PCR program is as follows:
    1. 95 degC for 1 min.
    2. 8 cycles as follows:
      1. 95 degC for 10 sec.
      2. 65 degC for 15 sec.
      3. 72 degC for 45 sec and acquire.
  6. Finish adapter PCR master mix (per sample) (this can be done during primer PCR):
    1. 35 ul 2X KAPA2G Robust Hot Start.
    2. 2 ul Adapter 1.
    3. 0.56 ul 50X SYBR Green.
    4. 30.44 ul H2O.
    5. Pause PCR program to add 68 ul adapter PCR master mix and 2 ul Adapter 2/barcodes.
    6. 95 degC for 1 min.
    7. 3 cycles as follows:
      1. 95 degC for 10 sec.
      2. 65 degC for 15 sec.
      3. 72 degC for 45 sec and acquire.
    8. 4 cycles as follows:
      1. 95 degC for 10 sec.
      2. 72 degC for 1 min and acquire.
    9. 72 degC for 2 min.
    10. 4 degC forever.
  7. Add 100 ul magnetic beads to each sample. Mix by pipetting and incubate 8 minutes off magnet.
  8. Place on magnet 5 minutes, then remove and discard supernatant.
  9. On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing.
  10. Let dry 10-15 minutes.
  11. Resuspend beads off magnet with 20 ul EB and incubate 2 minutes, then return to magnet for 1 minute.
  12. Extract supernatant and transfer to 0.2 ml tube.
  13. Run 3 ul on 6% PAGE, 20 minutes at 250 V. Use 3 ul loading dye per sample. Use 0.5 ul low mass ladder.
  14. In tip box lid, add 6 ul SYBR Gold to ~1 cm depth 0.5x TBE. Mix well before adding gels for staining. Incubate 2 minutes before imaging.