P-TABM-319 - P-TABM-319

5 ug of aRNA and 1 ul reference (green) spike mix (Amersham, Lucidea Universal ScoreCard, 63-0042-87), 1 ug of random nonamers (2ug/ul 9N, Microsynth), were heated at 70°C for 10 min then chilled on ice. The following ingredients were added: 4 ul First Strand Buffer, 2 ul 0.1M DTT, 1 ul of 10 mM dATP, 10 mM dGTP, 10 mM dTTP/2mM dCTP, 2ul Cy5-dCTP – depending on the sample or the dyeswap, 0.5 ul (10 units) of RNase Inhibitor (Invitrogen, 155 18-012), and 1.5 ul (300 units) SuperScript II Reverse Transcriptase (Invitrogen, 180 64-014) for a final volume of 20 ul. The reaction was incubated for 2 hours at 42°C before the RNA template was denatured with 2 ul 1M NaOH at 65°C for 10 min. The solution was immediately neutralized by adding 2 ul 1 M HCl plus 2 ul 3M NaAc pH 5.2 to assure an acidic pH for purification. The labeled cDNA was purified using a MinElute column (Qiagen, 28004). Manufacturer’s instructions were followed for the purification except the cDNA was eluted twice with 10 ul of the EB solution. The incorporation of the dyes was monitored using a Nanodrop spectrometer.
Experiment E-CAGE-189