Selected article for: "cdna template and reverse forward primer"
Author: SOMA, Takehisa; MATSUBAYASHI, Makoto; SASAI, Kazumi
Title: Detection of kobuvirus RNA in Japanese domestic dogs
  • Document date: 2016_8_2
    • ID: 5b8i71jd_6
    Snippet: CaKoV was detected by RT-PCR with a primer pair (Forward; 5′-CTCCCCTCAGCTGCCTTCTC-3, Reverse; 5′-GAGGATCTGAAATTTGGAAG-3′) that was amplified at nucleotide positions 7,368-7,619 (a partial 3D gene) of CaKoV 12D049 (KF924623) [4] , providing a 252-bp fragment, using a QIAGEN One Step RT-PCR kit (Qiagen science). Two point five µl of the extracted RNA was added to the 25 µl reaction mixtures containing 5 µl of 5 ×buffer (finally 1.5 mM MgC.....
    Document: CaKoV was detected by RT-PCR with a primer pair (Forward; 5′-CTCCCCTCAGCTGCCTTCTC-3, Reverse; 5′-GAGGATCTGAAATTTGGAAG-3′) that was amplified at nucleotide positions 7,368-7,619 (a partial 3D gene) of CaKoV 12D049 (KF924623) [4] , providing a 252-bp fragment, using a QIAGEN One Step RT-PCR kit (Qiagen science). Two point five µl of the extracted RNA was added to the 25 µl reaction mixtures containing 5 µl of 5 ×buffer (finally 1.5 mM MgCl 2 ), 0.4 mM of each deoxynucleozide triphosphate, 10 U RNase inhibitor (Promega, Madison, WI, U.S.A.), 0.8 µM of the primers and 1.0 µl of enzyme mix. The RNA was reverse transcribed at 50°C for 30 min, followed by inactivation of reverse transcriptase and denaturation of cDNA template at 95°C for 15 min. The cDNA was amplified in 35 sequential cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec and extension at 72°C for 40 sec, followed by a final extension of 72°C for 7 min.

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