Vapo protect mastercycler

PCR amplifications were performed using an Eppendorf vapo.protect Mastercycler (Eppendorf, Hamburg, Germany). Each 50 μl reaction contained 25 μl Promega PCR master mix (Promega, Southampton, UK), 1.0 μl each of primers Cc and Ac [10 mM] (La Fontaine et al., 1993 (link)) (Table 1), 2.5 μl of dimethyl sulfoxide (Fisher Scientific, Leicestershire, UK), 2 μl bovine serum albumin [10 mg ml⁻¹] (Sigma–Aldrich Ltd., Poole, Dorset, UK), 16.5 μl of nuclease free water and 2 μl of template DNA. For direct detection of D. nodosus, PCR was performed using Cc and Ac primers (La Fontaine et al., 1993 (link)) (Table 1) under the following conditions: 1 cycle of 95 °C for 2 min, 40 cycles of 95 °C for 1 min, 60 °C for 45 s and 72 °C for 2 min and a final extension step of 72 °C for 5 min. Samples that were negative using this approach, were tested further using nested PCR. In the first round 16S rRNA universal primers 27F and 1525R (Table 1) (Baker et al., 2003; Lane, 1991 ) were used in the conditions described above but with an annealing temperature of 55 °C, 1 μl of this product was used in the second round of PCR as described above. The PCR products were visualised under UV light.

To confirm expression of mRNA or DNA integration, PCR and RT-PCR were carried out. Genomic DNA was extracted from blood or cells using DNA extraction kit (DNeasy Blood&Tissue kit 69506, Qiagen, Limburg, Netherlands). Total RNAs were extracted using an RNA extraction kit (Easy spin total RNA extraction kit, Cat no. 17221, iNtRON, Seongnam-si, Korea). One ug total RNAs were used for synthesizing cDNA using a cDNA synthesis kit (RNA to cDNA EcoDry™ Premix Kit, PT5153-2, Clontech, California, US). Amplification of the target DNA using specific PCR primers was performed by PCR machine (Eppendorf Vapo Protect Mastercycler, Eppendorf, Germany).

Droplet digital PCR reactions consisted of 20 μL mixture per well containing 10 μL of ddPCR Probe Supermix (no deoxyuridine triphosphate, Bio-Rad, Hercules, CA, USA), 900 nM of primers, 250 nM of probe, and 5 μL of cDNA generated from either HIV-1 virion RNA or the ‘IVT’ standards. The ddPCR reactions were incorporated into droplets using the QX100 Droplet Generator (Bio-Rad). Nucleic acids were amplified with the following cycling conditions: 10 min at 95 °C, 45 cycles of 30 s at 95 °C and 59 °C for 60 s, and a final droplet cure step of 10 min at 98 °C using a Vapo.Protect Mastercycler^® (Eppendorf, Hamburg, Germany). Droplets were read and analyzed using Bio-Rad QX200 system and QuantaSoft software (version 1.7.4.0917) in ‘absolute quantification’ mode. Only wells containing ≥11,000 droplets were accepted for further analysis. No-RT controls (containing ≥10,000 cp HIV RNA) and no-template controls (NTCs) were included in every independent experiment.