The following thermal profile was used: a single cycle of reverse transcription for 30 min at 50 °C, 15 min at 94 °C for reverse transcriptase inactivation and DNA polymerase activation, followed by 30 amplification cycles of 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C.
The following thermal profile was used: a single cycle of reverse transcription for 30 min at 45°C, 2 min at 95°C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 amplification cycles of 15 sec at 95°C and 1 min at 60°C each (annealing-extension step).
The following thermal profile was used: a single cycle of reverse transcription for 15 min at 50°C, 2 min at 95°C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 amplification cycles of 15 sec at 95°C and 34 secs at 60°C each (annealing-extension step).
The neutralizing activity of antibodies was first determined using a single-cycle pseudovirus infection assay as previously described [26].
We first compared the ability of the two anti-HIV vectors to inhibit SHIV and also HIV-1 using a single-cycle infection assay.
Neutralization was evaluated using a single-cycle recombinant pseudotype virus assay by Monogram Biosciences, Inc. (San Francisco, CA) as described previously [14].
Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread.
We then used SPR to determine the binding affinities between these mutants and the Pikp-HMA using a single-cycle kinetics approach ([ Karlsson et al., 2006] Figure 4B, Figure 4 figure supplement 2B, Table 1), having confirmed a similar affinity of Pikp-HMA for AVR-PikD (Kd = 29 ± 3.5 nM) using this approach.
In our case we prepared 123 libraries which we sequenced using a single 600 cycle Miseq version 3 kit, with the Miseq machine parameters set to produce a separate fastq file containing index reads.
