Matt:LabNotes/2015-5-15

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Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set[edit]

  • Followed NEB E6300S protocol: [1]
  • BA8 Bulk Tissue RNA from Blue
    • 29 ng/ul
    • ~7ul total
  • Use 1ul with poly dT primer
  • Use 1ul with random nonamer (N9)
  • Use 1ul with poly dT primer and no reverse transcriptase as negative control
  • 2 tubes for poly dT
Components Volume
RNA 1 ul (30 ng)
d(T)23VN (50 µM) 2 ul
H2O 5 ul
Total 8 ul
  • 1 tube for N9
Components Volume
RNA 1 ul (30 ng)
N9 (100 µM) 2 ul
H2O 5 ul
Total 8 ul
  • Denatured RNA for 5 min at 70C and then put on ice
  • Added the following to tubes
    • 2ul H2O instead of Enzyme Mix for Negative Control (to test for DNA contamination in sample)
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • For N9 sample, incubate at 25C for 5min
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Stored in -20C over weekend
  • Purified cDNA in MinElute Qiagen PCR clean up column
    • Eluted 20ul