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