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]
- Add 2 ul HMW buffer to each white qPCR tube.
- Transfer samples to white qPCR tubes.
- Add 1 ul transpose (1:50 dilution) to each sample. Incubate 5 minutes at 55 degC.
- 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.
- Make the master mix for Exo- treatment: 1 ul Exo- and 1 ul dNTP per sample.
- 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]
- Finish primer PCR master mix (per sample):
- 15 ul 2X KAPA2G Robust Hot Start.
- 0.6 ul Orange primer.
- 0.6 ul Blue primer.
- 0.24 ul 50X SYBR Green.
- 0.56 ul H2O.
- Add 17 ul primer PCR master mix to each sample.
- Place tubes in real time thermocycler (Login: Eppendorf, Password: e).
- Use Background: EP_WWStriptubes, Wavelength: 520 SYBR, Probe: SYBR, 100 ul reaction volume.
- PCR program is as follows:
- 95 degC for 1 min.
- 8 cycles as follows:
- 95 degC for 10 sec.
- 65 degC for 15 sec.
- 72 degC for 45 sec and acquire.
- Finish adapter PCR master mix (per sample) (this can be done during primer PCR):
- 35 ul 2X KAPA2G Robust Hot Start.
- 2 ul Adapter 1.
- 0.56 ul 50X SYBR Green.
- 30.44 ul H2O.
- Pause PCR program to add 68 ul adapter PCR master mix and 2 ul Adapter 2/barcodes.
- 95 degC for 1 min.
- 3 cycles as follows:
- 95 degC for 10 sec.
- 65 degC for 15 sec.
- 72 degC for 45 sec and acquire.
- 4 cycles as follows:
- 95 degC for 10 sec.
- 72 degC for 1 min and acquire.
- 72 degC for 2 min.
- 4 degC forever.
- Add 100 ul magnetic beads to each sample. Mix by pipetting and incubate 8 minutes off magnet.
- Place on magnet 5 minutes, then remove and discard supernatant.
- On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing.
- Let dry 10-15 minutes.
- Resuspend beads off magnet with 20 ul EB and incubate 2 minutes, then return to magnet for 1 minute.
- Extract supernatant and transfer to 0.2 ml tube.
- 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.
- 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.