Matt:LabNotes/2015-1-12: Difference between revisions
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>Mzcai (Created page with "==Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 Probe Set== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis...") |
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* Universal Human Reference RNA: [[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA | Previously purified of contaminating DNA]] | * Universal Human Reference RNA: [[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA | Previously purified of contaminating DNA]] | ||
** About | ** About 2ul remaining from previous experiments | ||
** Estimate concentration ~0.5ug/ul | ** Estimate concentration ~0.5ug/ul | ||
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| align="center" style="background:#f0f0f0;"|''' Volume ''' | | align="center" style="background:#f0f0f0;"|''' Volume ''' | ||
|- | |- | ||
| RNA||1 | | RNA||1 ul (0.5 ug) | ||
|- | |- | ||
| d(T)23VN (50 µM)||2 ul | | d(T)23VN (50 µM)||2 ul | ||
|- | |- | ||
| H2O|| | | H2O||5 ul | ||
|- | |- | ||
| Total||8 ul | | Total||8 ul | ||
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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 | ||
*Purified cDNA in one Qiagen PCR clean up column and NTC in another | |||
**Eluted 30ul each and stored in -20C |
Latest revision as of 02:19, 13 January 2015
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