Matt:LabNotes/2015-1-12
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Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 Probe Set[edit]
- Followed NEB E6300S protocol: [1]
- Universal Human Reference RNA: Previously purified of contaminating DNA
- About 2ul remaining from previous experiments
- Estimate concentration ~0.5ug/ul
- 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components | Volume |
RNA | 1 ul (0.5 ug) |
d(T)23VN (50 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
Components | Volume |
M-MuLV Reaction Mix | 10 ul |
M-MuLV Enzyme Mix | 2 ul |
Total | 20 ul |
- 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