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====First Strand cDNA synthesis==== * Follow NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] Starting with 1ul of 1ug/ul total RNA brain reference * Mix components in microfuge tube {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (1 ug) |- | d(T)23VN (50 ΞΌM)||2 ul |- | H2O||5 ul |- | Total||8 ul |} * Denature RNA for 5 min at 70C and then put on ice * Add the following to tube {| {{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 |} * Incubate for one hour at 42C * Inactivate enzyme for 5 min at 80C * Store at -20C Lab Notes: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-3-8
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