Matt:LabNotes/2015-5-15: Difference between revisions
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>Mzcai |
>Mzcai m (→Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set) |
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* Incubated for one hour at 42C | * Incubated for one hour at 42C | ||
* Inactivated enzyme for 5 min at 80C | * Inactivated enzyme for 5 min at 80C | ||
*Stored in -20C over weekend | |||
*Purified cDNA in | *Purified cDNA in MinElute Qiagen PCR clean up column | ||
**Eluted | **Eluted 20ul |
Latest revision as of 02:45, 19 May 2015
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