Matt:LabNotes/2014-6-18

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Repeat Top48 Hexamer RT Primer in vitro Validation[edit]

Procedure[edit]

First Strand Synthesis[edit]

  • Mix RNA in Primer and H2O
  • Denature for 5 minutes at 70C
    • Put on ice right after
  • Add Reaction Mix and Enzyme
  • Incubate for 5 min at 25C
  • Incubate for 1 hour at 42C
  • Inactivate enzyme for 5 min at 80C
Random Primer Mix ' Oligo d(T)23 VN ' FISSEQ_RT ' Top48 Hexamer ' NTC ' No Enzyme '
Components Volume Components Volume Components Volume Components Volume Components Volume Components Volume
1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0 1.3ug/ul UHRR 0.5
Random Primer Mix (60uM) 2 Oligo d(T)23 VN (50uM) 2 FISSEQ_RT (100uM) 1 Top48 Hexamer (100uM) 1 Random Primer Mix (60uM) 2 Random Primer Mix (60uM) 2
H2O 5.5 H2O 5.5 H2O 6.5 H2O 6.5 H2O 6 H2O 5.5
Denature Step Denature Step Denature Step Denature Step Denature Step Denature Step
M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10
M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 H2O 2

Second Strand Synthesis[edit]

  • Add 48 ul H20
  • Add 8ul 10x Second strand synthesis reaction buffer
  • Add 4ul Second strand synthesis enzyme mix
  • Mix by pipetting
  • Incubate for 2.5 hours at 16C

Purification & Measure Concentration[edit]

  • Qiagen PCR Purification
    • Elute with 30ul
  • Nanodrop quant:
    • Random Primer Mix: 15.2 ng/ul
    • Oligo (dT)23 VN: 23.6 ng/ul
    • FISSEQ_RT: 9.7 ng/ul
    • Top48 Hexamer: 24.3 ng/ul
    • NTC: 14.5 ng/ul
    • No Enzyme: 25.0 ng/ul
  • Negative controls (NTC & No Enzyme) did not show significantly less DNA, could be RNA absorbing light and "fooling" the Nanodrop
  • Actual concentrations probably much less since previous comparison with Qubit showed 25x less

Nextera Tagmentation[edit]

  1. Add 2 ul HMW buffer to each PCR tube (8 total)
  2. Transfer 7ul of each sample to tubes
    • Random Primer Mix: 15.2 ng/ul
    • Oligo (dT)23 VN: 23.6 ng/ul
    • FISSEQ_RT: 9.7 ng/ul
    • Top48 Hexamer: 24.3 ng/ul
    • NTC: 14.5 ng/ul
    • No Enzyme: 25.0 ng/ul
    • NxtraPosControl (gDNA): 0.803 ng/ul
    • NxtraNegControl (H2O): 0 ng/ul
  3. Add 1 ul transposase (1:50 dilution) to each sample. Incubated 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

Adapter PCR[edit]

  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
    • Random Primer Mix: 26
    • Oligo (dT)23 VN: 27
    • FISSEQ_RT: 28
    • Top48 Hexamer: 29
    • NTC: 30
    • No Enzyme: 31
    • NxtraPosControl (gDNA): 32
    • NxtraNegControl (H2O): 33
    • NxtraPCRControl (H2O): 34
  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:06202014 Top48HexPrimer NxtraPCRtrial2.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:2014-06-20 RTPrimerinvitroTest Nextera trial2.jpg

  1. Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet
  2. Place on magnet 5 minutes, then remove and discard supernatant
  3. On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing
  4. Let dry 5 minutes
  5. Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute.
  6. Extract supernatant and transfer to 0.2 ml tube