From cbda55bc8ef8787c913e9051355ae1eff6fbf808 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Tue, 4 Mar 2025 16:07:32 +0100 Subject: [PATCH] added protocol and assay for MiniTn5forR401andR569 --- .gitattributes | 1 + .../README.md | 0 .../dataset/.gitkeep | 0 .../isa.assay.xlsx | Bin 0 -> 10631 bytes .../protocols/.gitkeep | 0 .../Mini-Tn5TransposonMutantInR401andR569.md | 3 +++ 6 files changed, 4 insertions(+) create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/README.md create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/isa.assay.xlsx create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md diff --git a/.gitattributes b/.gitattributes index 6608dce..7ca77f5 100644 --- a/.gitattributes +++ b/.gitattributes @@ -4,3 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diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md new file mode 100644 index 0000000..b4a2866 --- /dev/null +++ b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md @@ -0,0 +1,3 @@ +## Establishment of mini-Tn5 transposon mutant collections in R401 and R569 + +Mini Tn5-mutant collections of R401 and R569 were established similarly with only minor changes as described below. Liquid cultures of R401 or R569 and *E. coli* strain BW29427 carrying plasmid pUTmTn5Km2 (15) were grown overnight in no antibiotics or 25 μg/ml Kan and 50 μg/ml DAP at 25 °C or 37 °C, respectively. Conjugation was carried out as described above in *“Conjugation of E. coli and R401 and selection for first homologous recombination eventâ€*. For R401, the mating patch was taken up in 1 ml 50% TSB liquid medium and subsequently plated on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Zeocin in four different dilutions (undiluted, 1:3, 1:4, and 1:5) and left to grow at 25 °C for 48 h. Individual colonies were picked in 100 μl sterile 50% TSB in 96-well culture plates, sealed and left to grow at 25 °C and 180 rpm for 24 h. Subsequently, 100 μl 50% glycerol were added to each well and plates were frozen until further processing. The outer rows and columns were left uninoculated as to avoid positional effects in the subsequent forward genetic screen. For R569, resuspended mating patches were stocked at -80 °C in 700-μl aliquots using a final concentration of 25% Glycerol. 1:4 dilutions were plated onto 50% TSA plates supplemented with 120 μg/ml Kan, 50 μg/ml Rifampicin and 50 μg/ml Zeocin and incubated at 25 °C for 48h. Individual colonies were inoculated in 100 μl 50% TSB supplemented with the same antibiotics at the same concentrations in 96-well plates and incubated at 25 °C and 180 rpm for 48 h. Then, 100 μl 50% glycerol were added to each culture and plates were frozen at -80 °C. \ No newline at end of file -- GitLab