cDNAs were synthesized at 60°C in 20-µL reactions containing 200 nM TeI4c-MRF RT, RT buffer (75 mM KCl, 10 mM MgCl2, 20 mM Tris-HCl at pH 7.5, 1 mM DTT), 1 mM dNTPs, and 5 × 108 copies of 1.2-kb KanR RNA (Promega) with annealed primer P078 (5′-GGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAAC). After a 2-min preincubation in reaction medium containing all other components at 60°C, reactions were initiated by adding 1 mM dNTPs, incubated at 60°C for 30 min, and terminated by freezing on dry ice.
To quantitate KanR cDNA, 25-µL reactions were done in triplicate in 96-well plates with optical caps with each well containing 5 µL of cDNA (corresponding to 1.25 × 107 copies of kanR RNA, 2X TaqMan Gene Expression Master Mix [Life Technologies], primer-probe mix [200 nM FAM-BFQ1 probe], and 300 nM forward and reverse primers) Primer set 188–257: Forward P09 kan-188F, 5′-GGGTATAAATGGGCTCGCG; Reverse P030 kan-257R, 5′-CGGGCTTCCCATACAATCG; Taqman probe P031 kan-213T, 5′-(6FAM, 6-carboxyfluorescein)-TCGGGCAATCAGGTGCGACAATC/3IABkFQ/(Iowa Black Fluorescence Quencher). Primer set 562–634: Forward P001 kan-562F 5′-CGCTCAGGCGCAATCAC; Reverse P002 kan-634R 5′-CCAGCCATTACGCTCGTCAT; Taqman probe P003 kan-581T 5′-(6-FAM)-ATGAATAACGGTTTGGTTGATGCGAGTGA (TAMRA, tetramethyl-6-carboxyrhodamine) (de Rozieres et al. 2004 (link)). Plasmid pET9a (EMD Chemicals) was used to generate a standard curve to quantitate KanR cDNA levels. qPCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems), using the 9600 emulation mode protocol (50°C for 2 min, 95°C for 10 min, then 45 cycles at 95°C for 15 sec, and 60°C for 60 sec). Data were collected and analyzed by using Life Technologies SDS Versions 2.3 software, and cycle thresholds for cDNA samples were plotted against the standard curve to determine copy number equivalents.
To quantitate KanR cDNA, 25-µL reactions were done in triplicate in 96-well plates with optical caps with each well containing 5 µL of cDNA (corresponding to 1.25 × 107 copies of kanR RNA, 2X TaqMan Gene Expression Master Mix [Life Technologies], primer-probe mix [200 nM FAM-BFQ1 probe], and 300 nM forward and reverse primers) Primer set 188–257: Forward P09 kan-188F, 5′-GGGTATAAATGGGCTCGCG; Reverse P030 kan-257R, 5′-CGGGCTTCCCATACAATCG; Taqman probe P031 kan-213T, 5′-(6FAM, 6-carboxyfluorescein)-TCGGGCAATCAGGTGCGACAATC/3IABkFQ/(Iowa Black Fluorescence Quencher). Primer set 562–634: Forward P001 kan-562F 5′-CGCTCAGGCGCAATCAC; Reverse P002 kan-634R 5′-CCAGCCATTACGCTCGTCAT; Taqman probe P003 kan-581T 5′-(6-FAM)-ATGAATAACGGTTTGGTTGATGCGAGTGA (TAMRA, tetramethyl-6-carboxyrhodamine) (de Rozieres et al. 2004 (link)). Plasmid pET9a (EMD Chemicals) was used to generate a standard curve to quantitate KanR cDNA levels. qPCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems), using the 9600 emulation mode protocol (50°C for 2 min, 95°C for 10 min, then 45 cycles at 95°C for 15 sec, and 60°C for 60 sec). Data were collected and analyzed by using Life Technologies SDS Versions 2.3 software, and cycle thresholds for cDNA samples were plotted against the standard curve to determine copy number equivalents.
