Matt:LabNotes/2014-6-18

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Revision as of 18:32, 20 June 2014 by >Mzcai
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Repeat Top48 Hexamer RT Primer in vitro Validation

Procedure

First Strand Synthesis

  • 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

  • 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

  • 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

  1. Add 2 ul HMW buffer to each PCR tube (7 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 5.5 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