Cloneamp dna polymerase

The bivalent eNS2B₄₇NS3Pro was generated in the lab by adding a fragment with a stop codon, T7 promoter and IRES with another start codon. Firstly, the linear vector backbone was generated via PCR using CloneAmp™ DNA polymerase (Takara, Shiga, Japan). Subsequently, the linear backbone was purified using the Monarch^® PCR & DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Next, the fragment insertion was performed using In-Fusion^® HD Cloning Kit (Takara, Shiga, Japan) according to the manufacturer’s protocol. The transformed colonies were sub-cultured overnight in LB with appropriate antibiotics at 37 °C. Then, the plasmids were purified from transformed bacterial colonies using AxyPrep Plasmid Miniprep Kit (Corning, Corning, NY, USA) and sent for verification and Sanger sequencing (Bio Basic Asia Pacific Pte Ltd., Singapore). All of the primers used in the experiments are summarised in Table 2.

Open reading frames were amplified from cDNA prepared by reverse transcription of C. elegans total RNA using CloneAmp DNA polymerase (Takara Bio Inc) and the primers used are listed in Supplemental File 1. Amplicons were inserted into either pGBKT7 or pGADT7 cleaved with EcoRI and BamHI by In-Fusion cloning (Takara Bio Inc). The sequences were confirmed by Sanger sequencing (DNA sequencing and Services, Dundee University and Eurofins Genomics). pGADT7 and derivatives were transformed into yeast strain Y187 and pGBKT7 and derivatives into yeast strain Y2HGold (Takara Bio Inc.). Yeast transformation and subsequent mating of transformants were done using standard protocols. Protein-protein interactions were assayed by comparing growth on synthetic defined medium lacking leucine and tryptophan with growth on synthetic defined medium lacking in addition adenine and histidine.