Matt:LabNotes/2014-6-13

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Top48 Hexamer RT Primer in vitro Validation: Nextera Tagmentation[edit]

  • 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[edit]

Tagmentation[edit]

  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[edit]

  • 6-17-2014
  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.
    • PosCtrl: Indx 26
    • NegCtrl: Indx 27
    • Exp: Indx 28
    • gDNA: Indx 29
  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
    4. 31 ul H2O
  6. Add 70 ul of second round PCR master mix to each sample.
  7. 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.
  8. Incubate in thermocycler an additional 3 min @ 72 degC for final extension.

File:2014-06-17 Top48HexPrimer NexteraPCR.jpg

  1. 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.

File:2014-06-17 RTPrimerinvitroTest Nextera.jpg


Conclusion[edit]

  • Negative Control had double stranded DNA somehow nullifying the experiment