From f9979390e859e31f2c94e7f3ccc1de49a5dfe9fd Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Sat, 4 Nov 2023 16:08:33 +0100
Subject: [PATCH] update3

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 .../protocols/Quantitative real-time PCR (qRT-PCR).md           | 2 ++
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 create mode 100644 assays/measurements and quantification/protocols/Quantitative real-time PCR (qRT-PCR).md

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+## Quantitative real-time PCR (qRT-PCR)
+Cultures were sampled (0.5 ml) after 72 hours at the end of the growth experiment. The pellet was processed for RNA extraction using the PGTX method (dx.doi.org/10.17504/protocols.io.jm3ck8n, Pinto et al., 2009). The remaining DNA in the extracted RNA was removed by DNase digestion using the TURBO DNA-free™ (ThermoFischer) kit according to the manufacturer’s instructions. Extracted RNAs (250 ng) were used in a reverse transcriptase reaction using the RevertAid First Strand cDNA Synthesis Kit (ThermoFischer) according to the manufacturer’s instructions. The resulting cDNA was diluted 1:20. For performing qPCR, the DyNAmo ColorFlash SYBR Green qPCR Kit was used according to the manufacturer’s instructions. Primers for sqs, dxs and the housekeeping gene rpoA are shown in Table S2 (SI). Primer efficiencies were tested before performing qRT-PCR and were deemed sufficient to yield quantitative information (Figure S3; Table S3 (SI)). Changes in gene expression as fold changes compared to the control were determined using the 2−ΔΔCT method, using rpoA as a housekeeping gene and the Δshc strain subjected to the same rhamnose concentration as a control.
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