Faststart sybr green master mix with rox

For WITS experiments, 300 µl of tissue homogenate was inoculated into LB broth containing streptomycin (200 µg/ml) and kanamycin (40 µg/ml) as a recovery method to enrich for low abundance strains. An UltroSpec 2100pro spectrophotometer (Amersham Biosciences, Piscataway, NJ) was used to obtain optical density readings of the resulting bacterial cultures. Genomic DNA (gDNA) was extracted from 2×10⁹S. Typhimurium from each sample in duplicate using a DNeasy blood and tissue kit (Qiagen, 69506) as per the manufacturer's protocol for Gram-negative bacteria.
All qPCRs were performed on an Applied Biosystems 7300 real-time PCR system. A 25 µl reaction contained 12.5 µl of FastStart SYBR Green Master Mix with Rox (Roche, 04913914001), 8 µl DNase/RNase-free water, 0.75 µl of forward and reverse (10 µM) primers (Table S1), and 3 µl of gDNA (1–10 ng). Standard curves were generated using gDNA from each W1–W8 strain. Reaction conditions were 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; followed by a dissociation stage of 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s.

For RNA isolation from cultured cells, BMDMs were seeded (1x10⁶ cells per well of a 6-well plate) and infected in triplicate as described. Triplicate wells of infected macrophages were lysed in 1mL total of QIAzol (Qiagen). Total RNA was isolated from the aqueous phase using Econo-spin columns (Epoch Life Science) and then subjected to on-column PureLink DNase (Invitrogen) digestion. To generate cDNA, 2–4 μg total RNA was reverse transcribed using Maxima Reverse Transcriptase (Thermo Scientific), an oligo-dT primer, and pdN9 primers following manufacturer’s instructions. Quantitative PCR was performed on 1:10 to 1:50 dilutions of cDNA template using FastStart SYBR Green MasterMix with Rox (Roche). Reactions were run on an Mx3000P machine (Stratagene) and analyzed using MxPro software (Stratagene). Cycling parameters were as follows: 95°C for 10 min, followed by 40 cycles of 95°C (30 s), 55°C (60 s), and 72°C (30 s), followed by dissociation curve analysis. Abundances of CHOP and TRIB3 were normalized to HPRT levels.