Lb broth base

To verify HCV genotype and determine possible inter- and intra-patient subtype differences, HCV core amplicons (approximately 429 bp) were further analysed. A semi-nested PCR was performed with Sc2 and Ac2 as first-round primers and S7 and Ac2 as second-round primers [33 (link)] . The PCR conditions were as described for genotyping above. PCR products were purified from 2% agarose gel using QIAquick gel purification protocol (Qiagen Ltd., Germany) according to the manufacturer’s instructions. Purified amplicons were cloned directly into pCR 2.1-TOPO plasmid vector (~ 3.9 kb) and used to transform chemically competent Escherichia coli. Positive clones were detected through purification by Miniprep protocol (Qiagen Ltd.) and digestion with Eco RI. LB Agar, LB Broth Base, pCR 2.1 TOPO vector and Escherichia coli were obtained from Invitrogen, Life Technologies, Paisley, Scotland; and Eco RI was from Roche Diagnostics GmbH., Mannheim, Germany.
For each isolate, at least two clones were sequenced on both strands using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequencing products were purified by ethanol precipitation protocol. Electrophoresis and data acquisition were done on an automated ABI PRISM 310 genetic analyser (Applied Biosystems). Consensus nucleotide sequences obtained from the isolates were used in phylogenetic analysis.

Bacterial strains and plasmids used in this study are summarized in Table S1. Primers used in this study are summarized in Table S2. LB Broth Base and LB Agar (Invitrogen) were used to prepare medium for bacterial culture. Antibiotics were supplemented in the medium when necessary: tetracycline, 15 μg/mL; gentamicin, 20 μg/mL; kanamycin, 50 μg/mL; ampicillin, 100 μg/mL for E. coli DH5α, and tetracycline, 50 μg/mL; gentamicin, 50 μg/mL for P. aeruginosa PAO1.

The pcDNA3.1(+) plasmid was used as a template for inserting the MUC2-C (NCBI reference: MH593786.1^{8 (link)} (protein AZL49145.1) containing the amino acids 4356–5130 with an N-terminal IGk signal sequence, a 6 His-tag and the 3C precision cleavage tag in that order from the N-terminal. The mammalian expression vector was transformed through electroporation using XL1 Blue Electrocompetent cells (Agilent). The transformed cells were selected with the corresponding antibiotic and grown overnight in LB Broth Base (Invitrogen). DNA was prepared using PureLink™ HiPure Expi Plasmid Megaprep Kit (Thermo Fisher Scientific).