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In step 2, this product was used in a nested PCR with primers rPLU 3 (5'-TTT TTA TAA GGA TAA CTA CGG AAA AGC TGT-3') and rPLU Cal 2 (5'-CGC TAT TGG AGC TGG AAT TAC C-3') in a 25-[micro]L reaction under the conditions of 94[degrees]C (4 min) and 35 cycles at 94[degrees]C (10 see), 60[degrees]C (10 see), 72[degrees]C (45 see), with an additional extension at 72[degrees]C (4 min). ``It's an attempt to provide greater customer convenience to those golfers who want to walk,'' said GGA President Steven Boyers. JE-specific RNA was detected by using the Access quick one-step reverse transcription (RT)-PCR kit (Promega, Madison, WI, USA) with the primer pairs JED3S: ATG CGC GGA TCC GAC AAA CTG GCC CTG AA (1839-1867) and JED3C: GGG GAA GCT TCG TGC TTC CAG CTT TGT CC (2193-2165) on the basis of the sequence in domain III of the E gene of strain JaOArS982 (8). |
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