Matt:LabNotes/2014-6-13

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Top48 Hexamer RT Primer in vitro Validation: Nextera Tagmentation

  • Continued from: Matt:LabNotes/2014-6-9
  • Since running a gel without digesting RNA (eg with RNase A) can't confirm success of RT, must continue with Nextera, and successful tagmentation indicates successful RT and Second Strand Synthesis

Protocol

Tagmentation

  1. Add 2 ul HMW buffer to each PCR tube
  2. Transfer 7ul of each sample to tubes
    • PosCtrl: 1.06ng/ul
    • NegCtrl: 0.884ng/ul
    • Experimental: 0.818ng/ul
    • NexteraControl (gDNA): 0.803ng/ul
  3. Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5.5 minutes at 55 degC
  4. Dilute Qiagen 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 0.4 ul dNTP per reaction
  6. Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC

PCR with adapters and primers

  1. Finish first round PCR master mix (per reaction):
    1. 15 ul 2X KAPA fast SYBR.
    2. 0.6 ul Adapter 1.
    3. 1.4 ul H2O.
  2. Add 0.6 of barcode adapters to each sample.
  3. Add 17 ul of first round PCR master mix to each sample.
  4. Incubate in thermocycler:
    1. 95 degC for 1 min.
    2. 7 cycles:
      1. 95 degC for 10 sec.
      2. 62 degC for 15 sec.
      3. 72 degC for 45 sec.
  5. Finish second round PCR master mix:
    1. 35 ul KAPA fast SYBR
    2. 2 ul Primer 1
    3. 2 ul Primer 2
  6. Add 70 ul of second round PCR master mix to each sample.
  7. Place tubes in real time thermocycler (Login: Eppendorf, Password: e).
  8. Use Background: EP_WWStriptubes, Wavelength: 520 SYBR, 100 ul reaction volume.
  9. PCR program is as follows:
    1. 95 degC for 1 min.
    2. 10 cycles:
      1. 95 degC for 10 sec.
      2. 60 degC for 15 sec.
      3. 72 degC for 45 sec.
    3. Program can be stopped before 10 cycles (usually 7-8).
  10. Incubate in thermocycler an additional 3 min @ 72 degC for final extension.
  11. Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives.
    1. 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE.
    2. 25 min @ 250 V.
    3. Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe.
  12. Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet.
  13. Place on magnet 5 minutes, then remove and discard supernatant.
  14. On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing.
  15. Let dry 5 minutes.
  16. Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute.
  17. Extract supernatant and transfer to 0.2 ml tube. Proceed directly to size selection or store at 4 degC.