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==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-e6300] * Universal Human Reference RNA: [[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA | 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 {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (0.5 ug) |- | d(T)23VN (50 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 {| {{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 |} * 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
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