A synthetic gene was prepared by fusing 450 bp upstream and 450 bp downstream of the target gene (RSp0275, RSp0924, RSc0818, RSp0138, RSp0161, gspD, RSp1012, and RSp1007) (Eurofins Genomics). The fused gene was released from the provided vector by restriction enzyme digestion and introduced into the pK18mobsacB vector (Table S1). This plasmid was electroporated into RSSC cells (OD600 = 0.6, in 10% [wt/vol] glycerol), and the cells were incubated in SOC medium (1 mL, Sigma-Aldrich) for 6 h at 30°C. After this, kanamycin-resistant (50 μg/mL) and sucrose-sensitive (10% [wt/vol]) recombinants were selected. After 4 h of incubation in SOC medium, kanamycin-sensitive (50 μg/mL) and sucrose-resistant (10% [wt/vol]) recombinants were reselected. Finally, targeted deletions were confirmed by PCR. Both Δegl and ΔcbhA mutants were generous gifts from Hikichi (Kochi University) (71 (link)).
Soc medium
Manufactured by Merck Group
Sourced in United States
SOC medium is a type of nutrient-rich culture medium used for the growth and maintenance of bacterial cells, particularly during molecular biology and genetic engineering procedures. It provides the necessary nutrients and conditions for efficient bacterial growth and propagation.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Purelink hipure plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Neb 5 alpha competent escherichia coli cells, by New England Biolabs (1 mentions)
Top10 cells, by Thermo Fisher Scientific (1 mentions)
Bl21 de3 cells, by New England Biolabs (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
4 protocols using soc medium
1
Targeted Gene Deletion in RSSC Bacteria
2
Transformation of E. coli with Plasmid DNA
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NEB 5-alpha competent Escherichia coli cells (New England BioLabs, Hitchin, UK) were transformed with the vector according to the manufacturer’s instructions. Briefly, 10 ng of plasmid DNA was carefully mixed with cells and incubated on ice for 30 min. Cells were heat shocked at 42 °C for 30 s and chilled on ice for 5 min. After the addition of 950 µL of SOC medium (Sigma Aldrich, St. Louis, MO, USA), the mixture was shaken at 200 rpm for 60 min at 37 °C, and the cells were diluted and plated on selective LB agar (Invitrogen, Waltham, MA, USA) plates containing 100 µg/mL ampicillin (Gibco, Waltham, MA, USA). After incubation overnight at 37 °C, selected colonies were inoculated into 3 mL of fresh LB medium containing 100 µg/mL ampicillin and incubated overnight at 37 °C with shaking at 200 rpm. The vector construct with the RdRp sequence was verified by Sanger sequencing. Plasmids for subsequent transfection of lung cells were purified using a PureLink HiPure Plasmid Miniprep Kit (Invitrogen, Waltham, MA, USA).
3
Bacterial Strain Selection and Culture Conditions
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Standard DNA cloning was performed with chemically competent TOP10 cells (Invitrogen) and TG1 cells (Zymo Research). Combinatorial library cloning was performed with NEB 5-alpha electrocompetent cells or electrocompetent TG1 cells. Selection phage production was carried out with BL21(DE3) cells (NEB). All phage-assisted selection experiments and reporter assays were performed with TG1 cells. Genotypes of all strains are listed in Supplementary Table 3 . Cells were grown in Luria-Bertani medium (LB: 10 g l−1 Bacto-tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl), M9 minimal medium (6.8 g l−1 Na2HPO4, 3.0 g l−1 KH2PO4, 0.5 g l−1 NaCl, 1.0 g l−1 NH4Cl, 2 mM MgSO4, 100 μM CaCl2, 0.2% (w/v) glucose, 1 mM thiamine-HCl), 2 × TY medium (5 g l−1 NaCl, 10 g l−1 yeast extract, 16 g l−1 tryptone) or S.O.C. medium (Sigma-Aldrich). Chloramphenicol (25 μg ml−1), kanamycin (50 μg ml−1) and ampicillin (100 μg ml−1) were added where appropriate.
4
Methanogenic Archaea Diversity in Rumen
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Twenty of the 70 rumen samples (3 samples from each sample week, except 1 wk where only 2 samples were included) were selected to cover the entire transition period and from these, a mcrA gene clone library was constructed. To target the mcrA gene of all methanogens, we used the forward primer 5′-GGTGGTGTMG-GDTTYACHCARTA modified from Steinberg and Regan (2008) (link), and the reverse primer 5′-FAMCGTTCAT-BGCGTAGTTVGGRTAGT, used in the same study. Pooled PCR products for individual samples were purified with QIAquick PCR Purification Kit following the manufacturer's instructions (Qiagen GmbH). Purified PCR products from all the 20 samples were pooled at equimolar concentrations and subsequently ligated to pGEM-T Easy Vector according to the instruction of the manufacturer (Promega, Madison, WI). Plates were incubated overnight at 37°C, and positive clones were selected based on blue-white screening. Finally, white clones were transferred to microtiter-plates containing SOC medium (Sigma-Aldrich, St. Louis, MO) and sent for sequencing at MWG Eurofins (Ebersberg, Germany).
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