Hs qpcr sybr blue master mix

To confirm the expression of the inserted NA gene in bacterial DNA, we studied the expression of mRNA using real-time reverse transcriptase PCR (rRT-PCR) with NA-specific primers. Bacteria were grown in THB (Todd Hewitt Broth (Condalab, Madrid, Spain)) medium at 37 °C for 18 h. E. faecium L3-NA was cultivated with 5 μg/mL of erythromycin. For mRNA analysis, E. faecium L3 was collected at the logarithmic phase, when OD600 reached a value of 0.8–0.9, which corresponded to 3–5 × 10⁸ CFU/mL bacteria. Bacteria were washed three times in PBS by centrifugation at 3500 rpm for 20 min and suspended in PBS. Then, 10× concentrate was used for m RNA analysis. Isolation of total RNA was carried out using the GeneJET RNA Purification Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The isolated RNA was treated with 1 U/µL DNase (Invitrogen, Waltham, MA, USA), after which one-step rRT-PCR was performed in on a SFX96 thermocycler (Biorad, Hercules, CA, USA) using HS-qPCR SYBR Blue master-mix (Biolabmix, Novosibirsk, Russia). As a normalizing gene, we used D-alanine-D-alanine ligase gene of E. faecium L3 with the following primers: F-TTGAGGCAGACCAGATTGACG, R-TATGACAGCGACTCCGATTCC. Primers corresponding to the NA gene sequence were EV and FV (Table 1).

The expression of mRNA was studied using real-time PCR (rRT-PCR) with reverse transcriptase using primers specific for the S-protein. Bacteria were grown in THB medium at 37°C for 18 h. E. faecium L3-SARS was cultivated with 5 μg/ml of erythromycin (Sigma, United States). Bacteria were washed three times in PBS by centrifugation at 3,500 rpm for 20 min and suspended in PBS. There was 10x concentrate used for mRNA analysis. Isolation of total RNA was carried out using the GeneJET RNA Purification Kit (Thermo Scientific, Waltham, United States). The isolated RNA was treated with 1 U/µl DNase (Invitrogen, Waltham, United States) after which one-step rRT-PCR was performed on a SFX96 thermocycler (BioRad, Hercules, United States) using HS-qPCR SYBR Blue master-mix (Biolabmix, Novosibirsk, Russia). SarsS specific primers K1 и K2 were used for analysis of SarsS protein gene expression and D-alanine-D-alanine ligase gene of E. faecium L3 as follows: Dal1и Dal2 as the normalizing gene.

Genomic DNA was isolated from 30 to 50 flies according to the protocol reported earlier [48 ]. For detection of P-element-based transgene copy number by qPCR, we used the reference plasmid pP5′-Vps36-759bp-P3′ described previously [31 (link)]. The following three primer pairs were used: Vps36-realtime-F and Vps36-realtime-R specific for the Vps36 gene, qP5-F1 and qP5-R1 for the 5′ P-element end, and qP3-F1 and qP3-R1 for the 3′ P-element end (for primer sequences, see [31 (link)]). Importantly, primers qP5-F1 and qP5-R1 can detect only the “standard” 5′ P-element end of 585 bp in length, but not the minimal functional 5′ P-element end of 140 bp in length that is present in enhancer-trap Gal4 transposons. qPCR was performed with 100 ng of genomic DNA or 5 pg of the reference plasmid pP5′-Vps36-759bp-P3′, 400 nM of each primer in a 25-μl reaction mixture using the HS-qPCR SYBR Blue Master Mix (Biolabmix), and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) under the following conditions: incubation at 95 °C for 5 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Data analysis was performed using CFX Manager™ Software v3.0 (Bio-Rad). The 5′ and 3′ P-element copy numbers per diploid genome were calculated according to [31 (link)].