The In-Fusion HD Cloning Kit (Clontech, USA) was used to clone the VH, Vκ-Cκ and Vλ-Cλ genes into the pTT5 mammalian expression vector before transformation of Stellar Competent cells in a 96-well plate format (Clontech). Transformed cells were recovered in SOC medium (Clontech) with shaking at 37 °C for 45–60 min before plating out onto LB agar plates (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 1.5% (w/v) agar) containing 100 µg/ml ampicillin. Single colonies picked for inoculation of 2xTY media containing 100 µg/ml ampicillin in a Greiner deep well, 96-well plate (Sigma). Plasmid miniprep DNA was isolated from cultures in 96-well microtitre plates and an epMotion® 5075 laboratory robot (Eppendorf, Germany) and stored at –20 °C until required for mammalian transfections following sequence analysis of complementary determining region (CDR) diversity and comparison to germline sequences.
Soc medium
Manufactured by Takara Bio
Sourced in United States
SOC medium is a rich growth medium used for culturing bacterial cells, particularly Escherichia coli, during molecular biology experiments. It provides essential nutrients and supports rapid cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
In fusion hd cloning kit, by Takara Bio (1 mentions)
Elepo21, by Nepa Gene (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Dna ligation mighty mix, by Takara Bio (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Stellar competent cells, by Takara Bio (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Nucleospin plasmid miniprep kit, by Macherey-Nagel (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
Lb medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using soc medium
1
Scalable Recombinant Antibody Production
2
Culturing Haloarchaea and E. coli
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All strains and plasmids used in this study are listed and described in Table 1 . Strains of H. volcanii were grown at 42 °C while shaking at 200 rpm using either rich medium (Hv-YPC) or selective rich medium (Hv-Ca) developed by Allers et al. [41 (link)] and outlined in the Halohandbook [42 ]. For ∆pyrE2 strains, media was supplemented with uracil (50 µg/mL) and 5-fluoroorotic acid (50 µg/mL) as needed. Strains of E. coli were grown at 37 °C while shaking at 200 rpm in either Lysogeny Broth (LB) or S.O.C. medium (Clontech, Mountain View, CA, USA). Ampicillin (100 µg/mL) and X-gal (20 µg/mL) were added to the media when needed.
3
Synthetic Phage Production in E. coli
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The assembled genomes (10 μl) were electroporated into E. coli HST08 Premium Electro-Cells (Takara Bio Inc., Shiga, Japan) with ELEPO21 (Nepa Gene Co., Ltd., Chiba, Japan; poring pulse [voltage: 1500 V, pulse length: 2.5 ms, pulse interval: 50 ms, number of pulses: 1, polarity: +], transfer pulse [voltage: 150 V, pulse length: 50 ms, pulse interval: 50 ms, number of pulses: 5, polarity: +/−]). After electroporation, samples were added to 1 ml SOC medium (Takara Bio Inc.) and incubated at 37 °C for 20 to 60 min with shaking at 200 rpm. The samples (500 μl to 1 ml) and E. coli O157 strain ATCC 43888 overnight cultures (200–300 μl) were added to 3 ml of LTA. They were poured onto LB plates and incubated at 37 °C overnight. The plaques were counted, and synthetic phages were kept in 100 μl of SM buffer at 4 °C for the following experiments.
4
Evaluating tmexCD1-toprJ1 Role in Tigecycline Resistance
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To confirm the role of tmexCD1-toprJ1 in tigecycline resistance, a 7,205 bp full-length ORF of tmexCD1-toprJ1 was amplified by PCR (Table S1). Subsequently, 0·3 pmol of purified PCR product was incubated with 1 μL T-Vector pMD-19 (TaKaRa) and 5 μL DNA Ligation Mighty Mix (TaKaRa) at 16°C overnight. The recombinant plasmid pUC19-tmexCD1-toprJ1 was then transformed into competent E. coli DH5α by incubating on ice for 30 min followed by incubating for 45 s at 42°C. Immediately thereafter, the DH5α was transferred to SOC medium (TaKaRa) at 37°C for one hour and then was coated on Luria–Bertani Agar (LB) medium containing 100 μg/ml of ampicillin at 37°C overnight. Visible strains after overnight were verified by PCR and then subjected to susceptibility testing using broth microdilution method. The recombinant vector was then extracted and transferred into K. pneumoniae ATCC 13883 via electroporation.
5
Cloning and Validation of RAC1P29S
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pBMN-FKBP-YFP destabilization domain plasmid was purchased from Addgene RRID:Addgene_31763. Human RAC1P29S cDNA was cloned into the vector using EcoRI and XhoI restriction enzymes (New England Biolabs) and ligated using T4 ligase (New England Biolabs). Transformations were performed in Stellar competent cells (Takara). 2 μL of reaction was added to 50 μL Stellar competent cells on ice for 30 mins followed by 45 second heat shock at 42 degrees C and resuspended in 500 μL SOC medium (Takara) and placed 1 hour shaking 200 rpm at 30 degrees C. 200 μL bacteria was then plated onto LB/AMP plate and incubated overnight at 30 degrees C. Colonies were mini-prepped with NucleoSpin Plasmid mini prep kit (Machery-Nagel). Sequences were verified by Genewiz (Azenta) plasmid sequencing services with primer sequence 5’ – TTC TGG AGC GGC GAG CAT GCA TGG AAC AGA AAC TCA TCT CTG AAG AGG - 3’. The destabilization domain was removed from the plasmid via restriction enzymes BamHI and EcoRI (New England Biolabs) for constitutive expression in the YUMM1.7 cell lines.
6
Recombineering of Bacterial Artificial Chromosome
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The BAC vector p17-9A containing the miu cluster was purified using the QIAGEN Plasmid Midi Kit (Qiagen). Approximately 200 ng of the BAC p17-9A was transformed into 70 µL of electrocompetent E. coli SW105 cells [33 (link)] in a 1-mm cuvette (NEPAGENE, Ichikawa, Chiba, Japan) at 1,800 V for one pulse. E.coli transformants were recovered at 30 °C for 1 h in 1 mL of SOC medium (Takara Bio) and then plated onto LB medium (Thermo Fisher Scientific) containing chloramphenicol at 30 °C for 24–36 h. Individual colonies were picked up and verified by colony PCR.
The Red/ET recombination modification cassette 5TA-KanR was amplified using the primer pair ploTA-Kan red F/R from the vector p5TA-KanR. Approximately 200 ng of the modification cassette “5TA-KanR” was transformed into electrocompetent E. coli SW105 cells harboring the BAC vector p17-9A via electroporation followed by recovery and cultivation under the above-mentioned conditions to obtain the recombinant miu BAC harboring the complete miu cluster and 5.0 kbp M. xanthus homologous fragment (5TA). The resulting colonies were verified by PCR using the two primer pairs loTA cF/R and kanf/pCC1BAC R (Figure S1 ).
The Red/ET recombination modification cassette 5TA-KanR was amplified using the primer pair ploTA-Kan red F/R from the vector p5TA-KanR. Approximately 200 ng of the modification cassette “5TA-KanR” was transformed into electrocompetent E. coli SW105 cells harboring the BAC vector p17-9A via electroporation followed by recovery and cultivation under the above-mentioned conditions to obtain the recombinant miu BAC harboring the complete miu cluster and 5.0 kbp M. xanthus homologous fragment (5TA). The resulting colonies were verified by PCR using the two primer pairs loTA cF/R and kanf/pCC1BAC R (
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