Superscript 3 one step pcr kit

The optimized protocol started with the HIV viral RNA isolation from patients' plasma specimens using a spin column-based QIAamp Viral RNA extraction kit (Qiagen) as per the manufacturer's instructions [14 ]. The initial input volume of plasma specimen was 400 μL and the RNA extracts were recovered using an elution volume of 60 μL. Viral RNA extraction was followed by one-step RT-PCR using Invitrogen Superscript III One-step PCR kit (Invitrogen, Canada) with Platinum High Fidelity Taq (Invitrogen, Canada). The RT-PCR primers are described in Table 1 and the derived amplicon encompasses nucleotides 2,071~3,370 of HIV-1 pol gene as referring to HIV-1 HXB-2 sequence (GenBank Accession number: K03455) [14 , 15 ]. The 50 μL RT-PCR reaction contains 1x SuperScript III reaction buffer, 1.6 mM MgSO₄, forward and reverse primers at 200 nM each, 1 μL SuperScript III enzyme mix, and 12 μL of RNA extracts. The RT-PCR was performed on a GeneAmp PCR system 9700 thermocycler (ABI, USA) with cycling conditions as follows: (1) an initial reverse transcription step of 30 min at 50°C; (2) 2 min at 94°C for DNA denaturation and Taq DNA polymerase activation; (3) 40 cycles of 20 s at 94°C for DNA melting, 30 s at 50°C for annealing, and 1.5 min at 68°C for elongation; (4) a final extension step of 5 min at 68°C; and (5) 4°C till the end.