Matt:LabNotes/2016-6-4

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Make First Strand cDNA for in vitro Capture with TB12k_Apr2016 Probe Set

  • Followed NEB M0368 protocol: [1]
  • BA8 Bulk Tissue RNA from Blue
    • 29 ng/ul
    • ~4ul total
  • Use 1ul with random nonamer (N9)
  • Use 1ul with no reverse transcriptase as negative control
  • 2 tubes
Components Volume
RNA 1 ul (30 ng)
N9 (100 µM) 2 ul
10 mM dNTP 1 ul
H2O 6 ul
Total 10 ul
  • Denatured RNA for 5 min at 65C and then put on ice
  • Added the following to tubes
    • 1ul H2O instead of ProtoScript II RT for Negative Control (to test for DNA contamination in sample)
Components Volume
5X ProtoScript II Buffer 4 ul
0.1 M DTT 2 ul
ProtoScript II RT (200 U/µl) 1 ul
RNase Inhibitor (40 U/µl) 0.2 ul
H2O 2.8 ul
Total 20 ul
  • Incubate at 25C for 5min
  • Incubated for one hour at 42C
  • Inactivated enzyme for 20 min at 65C
  • Stored in -20C over weekend
  • Purified cDNA in MinElute Qiagen PCR clean up column
    • Eluted 20ul