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
- Followed NEB E6300S protocol: [1]
- BA8 Bulk Tissue RNA from Blue
- 29 ng/ul
- 4ul 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
- Purified cDNA in one Qiagen PCR clean up column and NTC in another
- Eluted 30ul each and stored in -20C