100 bp dna ladder

Genomic DNA was extracted from fresh 4-week-old bacterial cultures grown on L-J medium as previous reported (Pang et al., 2012 (link)). Bacterial colonies were harvested from solid cultures and were transferred into microcentrifuge tubes containing 500 μL Tris-EDTA (TE) buffer. After inactivation in a 100°C water bath for 30 min, supernatants containing DNA templates were subjected to PCR amplification. The classical 24-locus MIRU-VNTR method was performed to genotype MTB isolates (Supply et al., 2006 (link)). To detect differences in repeat numbers, PCR products were examined by standard agarose gel electrophoresis (1.5% agarose), with visualization of DNA by fluorescence using GelRed dye^®. The 100-bp DNA ladder (Transgene, Beijing, China) was loaded into wells of every fourth lane to serve as a size marker. Amplicon sizes were estimated using Quantity One software (Bio-Rad, Hercules, CA, United States). A reinfection case was defined by the occurrence of strains with different 24-locus MIRU-VNTR patterns at two or more loci between first and second TB episodes, as previously reported (Martín et al., 2011 (link); Pang et al., 2015 (link)).

Both PCR assays were performed in a 20μL final reaction volume, which consisted of 10 μL 2xEasyTaq PCR superMix (TransGene Biotech; contained EasyTaq DNA polymerase, dNTPs, and optimised buffer), 3 μL of DNA template, 0.8 μL of each forward (HMT-F) and reverse (HMT-R) primer for amplification of a partial sequence of 18S rRNA, and of LITSR and L5.8S primers for ITS1. The volume of the reaction was completed with the addition of nuclease free water. PCR mixtures were spun down briefly (5–10 s), then placed in a thermal cycler (TCY, Crealcon, NL) and subjected to the following cycling conditions: initial denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 40 s, and a final extension step at 72 °C for 8 min.
The amplified DNA fragments were visualised via 1.5% agarose gel electrophoresis, using prime save dye (GeneAid) in TBE buffer at 100 V for 60 min at room temperature. Gels were photographed after electrophoresis and amplicon size was determined by comparison with a 100-bp DNA ladder (TransGene Biotech).

PCR products were separated by 2% agarose gel electrophoresis with TAE buffer using a 100 bp DNA ladder (TransGen Biotech, Beijing, China). The bands were photographed under an Azure C600 imager (Azure Biosystems, Dublin, CA).