Phusion hot start 2 high fidelity pcr master mix

The enriched cells by FACS were incubated in a 95°C block heater for 10 min and the sample was used as a template for PCR amplification with the primers of CDR3-forward: 5′-ACACCGCCATCTACTACTGC-3′ and CDR3-reverse: 5′-GCTGCTCACTGTCACTTGTG-3′. Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific) was used according to the manufacture’s instruction. Up to 500 cells were used as template in 50 μl reaction mixtures containing 0.5 μM of each primer. The PCR procedure is 98°C for 60 s, 50 cycles of 98°C for 30 s, 62°C for 30 s, and 72°C for 30 s, and a final 10-min extension at 72°C. PCR products were used for the next round of cloning into sdAb vector followed by transforming DH5α or for CDR3 sequencing of individual clones. The sequencing primer is 5′-GCACCAAAATCAACGGGAC-3′. After high-throughput sequencing, docking of the recovered sdAbs and SQSTM1 molecules was conducted using HDOCK as described previously (Yan et al, 2020 (link)).

Total DNA was extracted from cells using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. An MT-ND4 gene fragment spanning the SNP site in HeLa and 293T cells was amplified with the Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific). The primers used for the PCR are listed in Supplementary Table 3. PCR products were purified with the QIAquick PCR Purification Kit (Qiagen) and subjected to Sanger sequencing at Azenta.

PCR reactions (50 µL) contained 25 µL Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific, USA), 0.2 µM of each primer (Invitrogen, Thermo Scientific, NZ), 0.05 µg template gDNA, and an appropriate volume of PCR-grade H₂O. Optimised PCR cycling conditions: initial denaturation at 98 °C for 30 s; 30 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 3 min 24 s; and final extension at 72 °C for 10 min.

Platinum™ SuperFi™ II PCR Master Mix (Thermofisher Scientific, Melbourne, Australia) and Hot Start II High-Fidelity PCR Master Mix (Thermofisher Scientific, Melbourne, Australia) were used to perform FABP4 and SCD PCR amplification assay under the same PCR conditions described below. The amplification reactions were performed in a total volume of 50 µL containing 25 µL of 2× Platinum™ SuperFi™ II PCR Master Mix or Phusion Hot Start II High-Fidelity PCR Master Mix (Thermofisher Scientific, Melbourne, Australia), 0.5 µM of each primer, and 100 ng of DNA template. PCR was performed in a SimpliAmp™ Thermal Cycler (Thermofisher Scientific, Melbourne, Australia), in a three-step protocol, using the following conditions: 98 °C initial denaturation in 1 min (1 cycle); 98 °C denaturation for 15 s; 60 °C (FABP4)/and 65 °C (SCD) annealing for 15 s; 72 °C extension for 9 min; 72 °C final extension for 9 min; 4 °C hold for 35 cycles. PCR success was checked in a 0.8% agarose gel electrophoresis image, as depicted in Figure 2 and Figure 3.