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==RT w random hexamer== *Dilute FISSEQ_RT primer from 100uM to 10uM *UV mineral oil, 0.2ml tube in hood {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="216" height="16" | Content | width="65" | Volume |- style="font-size:12pt" align="center" |style="background-color:#EBF1DE" height="16" | 25 mM dNTP (Enzymatics) | align="center" | 1 |- style="font-size:12pt" align="center" |style="background-color:#EBF1DE;color:#333333" height="16" | 2µg Total RNA | align="center" | 4.2 |- style="font-size:12pt" align="center" |style="background-color:#EBF1DE" height="16" | 10uM FISSEQ_RT (final 10 µg/mL) | align="center" | 1.34 |- style="font-size:12pt" align="center" |style="background-color:#EBF1DE" height="16" | 4mM aminoallyl dUTP (Anaspec) | align="center" | 1 |} *Heat reaction for 5 minutes at 65°C. *Spin briefly (5 sec) to pull down condensate and place immediately on ice. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="216" height="16" | Content | width="65" | Volume |- style="font-size:12pt" align="center" | height="16" | RNase Inhibitor (Enzymatics) | align="center" | 0.4 |- style="font-size:12pt" align="center" | height="16" | 10x MMLV reaction buffer | align="center" | 1 |- style="font-size:12pt" align="center" | height="16" | ddh2o | align="center" | 1.06 |} *2 minutes @ 25°C *Add 1 µL (200 units) M-MuLV Reverse Transcriptase and mix by gently pipetting sample *Add mineral oil to prevent evaporation *25C 10min *Incubate at 42°C overnight *Inactivate enzyme at 85°C for 10 minutes. *Store products at -20°C *save 1ul for denaturing gel
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