Gel logic 440

MLP was characterized using SDS-PAGE using standard procedures [13 (link)]. Briefly, protein samples (5 μg/well) were separated under reducing conditions (2-mercaptoethanol, heated for 5 min at 100 °C) or under non-reducing conditions (without 2-mercaptoethanol and heating) in SDS-PAGE, performed on a Bio-Rad microprotein electrophoresis system (BioRad Laboratories, Inc., Hercules, CA, USA) using 12% separation gel and 5% stacking gel, then stained with colloidal blue G250 (Sigma, Saint Louis, MO, USA) [14 (link)]. The gel was then scanned with the Kodak Gel Logic 440 imaging system, and the density of pixels of the primary band at the appropriate molecular weight in reducing conditions was compared to the total density of all protein bands in the sample lane.

Plaque sizes for 13 HRM genotypes were estimated from digital photographs of plaques formed on ERA. All LB plates used for plaque assays were poured at the same time and were weighed to maintain consistency. Each mutant's lysate was diluted and plated such that between 20 to 100 plaques formed on the ERA lawn after 48 hrs growth. Digital photographs were taken using a Kodak Gel Logic 440 digital imaging system. ImageJ software (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/) was used to estimate the total area of the plaque. For each genotype, at least 35 plaque size estimates were made across 3 plates.

Two HCV genomic regions, Core/E1(C/E1) and NS5B, were amplified by nested-PCR with 4 pairs of target-specific primers [9 (link)] (for details, see Table S1). PCR was conducted with a total volume of 50 μL, which contains 5 μL of 10× Ex Taq buffer (20 mM Mg²⁺ plus), 4 μL of dNTPs mixture (2.5 mM each), 0.25 μL of TaKaRa Ex Taq (5U/μL), 1 μL of 1U TaKaRa Ex Taq HS DNA Polymerase, Outer primer (10 μM), 1 μL of Inner primer (10 μM), and 1 μL of cDNA. The final volume was added up to 50 μL with sterilized distilled water. After activation of the Taq polymerase at 95 °C for 10 min, PCR was carried out in a AB7300 thermal cycler (Applied Biosystems, Foster City, CA, USA) for 40 cycles, each cycle consisting of denaturation at 95 °C for 1 min, annealing at 48 °C (C/E1)/50 °C (NS5B) for 1 min, and extension at 72 °C for 1 min, followed by a final 5 min extension at 72 °C. A HCV positive control and negative control (water without DNA) were included in each batch of PCR reactions and amplification. The PCR products were then loaded to a 1% agarose gel, which were stained with ethidium bromide and visualized under UV illumination Image system (Kodak Gel logic 440, Rochester, NY, USA). The expected size of the PCR amplified C/E1 and NS5B products were 468 and 388 bp, respectively (a representative image showing results of the agarose gel electrophoresis can be found in Figure S1).