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==Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 + suppressor oligo v2 Probe Set== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] * 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 {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (30 ng) |- | d(T)23VN (50 Β΅M)||2 ul |- | H2O||5 ul |- | Total||8 ul |} *1 tube for N9 {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (30 ng) |- | N9 (100 Β΅M)||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) {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' 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
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