Superscript 3 first strand synthesis kit for rt pcr

Strains were cultured as described above and 1 mL of culture was collected at an OD₆₀₀ of 1. Cells were pelleted and washed with 1X PBS and RNA was isolated using the RNeasy Plus Kit (Qiagen) according to the manufacturer’s instructions. Modifications were made to the protocol as described earlier [15 (link)]. cDNA was synthesized using the Superscript III First-Strand Synthesis for RT-PCR kit (Invitrogen; Eugene, OR), according to the manufacturer’s instructions. DNA contamination was tested by performing cDNA synthesis in the absence of reverse transcriptase; this control indicated the absence of DNA in our RNA samples (not shown). Quantitative Real-time-PCR (qRT-PCR) was performed as previously reported using the LightCycler 480 Instrument (Roche) [15 (link)]. Primers exsARTfor and exsARTrev [8 ] were used to amplify exsA from nucleotides 436 to 676. Samples were normalized to the fbp transcript using primers PA5110for and PA5110rev, as described [15 (link)]. For semiquantitative RT-PCR, cDNA was subjected to standard PCR with primer pairs QpolBfor/QpolBrev and P5110for/5110rev [10 (link)], with the following thermocycler conditions: 95°C 5 min., 25X(95°C 1 min., 55°C 30 sec., 72°C 1 min.), 72°C 10 min. PCR reaction was then electrophoresed through a 1% agarose gel.

cDNA synthesis was performed using the Invitrogen Superscript III First Strand Synthesis for RT-PCR kit, with Random Hexamer primers, according to the manufacturer’s instructions. Quantitative PCR was performed using the Biorad iQ SYBR Green Supermix on a CFX96 Real Time PCR Detection System, with the following gene-specific primers: mouse GAPDH 5’-catgttccagtatgactccactc ; mouse GAPDH 3’-ggcctcaccccatttgatgt mouse Mena 5’-gggcagaaagattcaagacc ; mouse Mena 3’-gcgaagacattggcatcc mouse Dyrk1a 5’-caaacggagtgcaatcaaga ; mouse Dyrk1a 3’-agcacctctggagaccgata mouse Robo1 5’-catcaagaggatcagggagc ; mouse Robo1 3’-ggttgtcttcagctttcagtttc mouse Elavl1 5’-agccaatcccaaccagaac ; mouse Elavl1 3’-acaccagaaatcccactcatg mouse β-Ctnn 5’-ctatcccagaggctttatccaag ; mouse β-Ctnn 3’-ccagagtgaaaagaacggtagc mouse Khsrp 5’-gccaatcagactacaccaagg ; mouse Khsrp 3’-gccacttgtgttgcttcttg mouse Eif4ebp2 5’-ccatctgcccaatatccctg ; mouse Eif4ebp2 3’-tgtccatctcaaactgagcc mouse Vamp2 5’-aagttgtcggagctggatg ; mouse Vamp2 3’-cgcagatcactcccaagatg