Miniprep protocol

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.

Yeast cells containing scFv outputs selected on CD2KA and USP11 and yeast transformed with the same outputs cloned as scTGP were grown and induced. Antigen staining was performed at 100 nM. Washes and secondary fluorescent reagent incubations were carried out as described above. Yeast containing outputs in the scTGP format were further enriched by yeast sorting using the green fluorescence signal to assess display levels. The sorted yeast libraries were cultured in SD/CAA media and a modified Qiagen miniprep protocol was used to prepare plasmids from yeast cells. In this protocol, yeast cell walls are disrupted by vortexing at high speed for 10 minutes with acid washed beads (Sigma Inc. #G8772) and double the amount of P1 and P2 buffers are added. The manufacturer’s instructions were followed for the remainder of the protocol and the yeast plasmid DNA was used in subsequent sequencing reactions. The yeast plasmid DNA was also used to transform Omnimax T1 cells to obtain single colonies for Sanger sequencing of scTGP clones.