All experiments were performed in E. coli TG1 cells [F′ traD36 lacIq Δ(lacZ) M15 pro A+B+/supE Δ(hsdM-mcrB)5 (rk− mk− McrB-) thi Δ(lac-proAB)]. Plasmid combinations were transformed into chemically competent TG1 cells, plated on LB + Agar plates containing 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol, and incubated overnight ∼17 h at 37°C. The plates were taken out of the incubator in the morning and left at room temperature for ∼7 h at which point three colonies were picked to inoculate 300 µL of LB containing carbenicillin and chloramphenicol at the concentrations above in a 2-mL 96-well block (Costar 3960). Cultures were grown overnight, ∼17 h at 37°C at 1000 rpm in a Labnet Vortemp 56 benchtop shaker. Four microliters of this overnight culture were then added to 196 µL (1:50 dilution) of supplemented M9 minimal media (1×M9 minimal salts, 1 mM thiamine hydrochloride, 0.4% glycerol, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2) containing the antibiotics above and grown for 3 h at the same conditions as the overnight culture. One hundred microliters of this culture were then transferred to a 96-well plate (Costar 3631) containing 100 µL of phosphate buffered saline (PBS). Fluorescence (FL) (485 nm excitation, 520 nm emission) and optical density (OD 600 nm) were measured using a Biotek Synergy H1M plate reader.
Vortemp 56
Manufactured by Labnet
Sourced in United States
The Vortemp 56 is a vortex mixer designed for mixing and resuspending samples in various laboratory applications. It features a compact size, variable speed control, and a stable platform for secure sample handling.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1m, by Agilent Technologies (1 mentions)
Quantigene plex assay kit, by Thermo Fisher Scientific (1 mentions)
Waterbath b 480, by Büchi (1 mentions)
Synergy h1 plate, by Agilent Technologies (1 mentions)
Wallac dbs puncher, by PerkinElmer (1 mentions)
Robotic liquid handler, by Tecan (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
14 protocols using vortemp 56
1
High-throughput E. coli Transformation Assay
2
In-cell SHAPE-Seq Protocol for E. coli
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All in-cell SHAPE-Seq experiments were performed in E. coli TG1 cells. Plasmids were transformed into chemically competent TG1 cells plated on LB + Agar plates containing 100 µg/mL carbenicillin or 34 µg/mL chloramphenicol and incubated overnight, ∼17 h at 37°C. The plates were taken out of the incubator in the morning and left at room temperature for ∼7 h at which point, colonies were picked to inoculate 500 µL of LB + antibiotic at the concentrations above in a 2 mL 96-well block (Costar 3960). Cultures were grown overnight, ∼17 h at 37°C at 1000 rpm in a Labnet Vortemp 56 benchtop shaker. Twenty-four microliters of this overnight culture were then used to subculture into 1.2 mL of LB + antibiotic. The subculture was grown under the same conditions for three hours before performing structure probing.
3
Quantifying mRNA expression from tissue samples
4
Doxorubicin-Loaded Liposome Preparation
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Liposomes were prepared by the film-hydration method combined with a pH gradient for active drug loading [30 (link), 38 (link), 52 (link)].
Briefly, HSPC:CH:DSPE-PEG2000 at a molar ratio of 1.85:1:0.12, were dissolved in a solution of chloroform:methanol [9:1 (v/v)]. Organic solvents were removed by rotary evaporation (Büchi Waterbath B-480, Switzerland) at 65 °C obtaining the film, which was hydrated with ammonium sulphate (250 mM) at pH 5.5 under constant stirring. The liposomal solution was extruded five times through a polycarbonate membrane with a drain disk. To obtain a homogeneous population of liposomes, membranes with different pore size were used, 200 nm, 100 nm and 80 nm.
Ammonium sulphate buffer was exchanged with Hepes saline buffer (Hepes 10 mM, EDTA 5 mM and NaCl 150 mM; pH 6.7) through a PD-10 column and LPs were incubated with Dox at a molar ratio of 0.1:1(Dox:Lipid) in a thermoshaker (Vortemp 56, Labnet, USA) for 1 h at 60 °C. Non-encapsulated Dox was removed by ultracentrifugation at 40,000 rpm for 2 h at 4 °C and the final Dox liposomal formulation was kept in Hepes saline at 4 °C until use.
Briefly, HSPC:CH:DSPE-PEG2000 at a molar ratio of 1.85:1:0.12, were dissolved in a solution of chloroform:methanol [9:1 (v/v)]. Organic solvents were removed by rotary evaporation (Büchi Waterbath B-480, Switzerland) at 65 °C obtaining the film, which was hydrated with ammonium sulphate (250 mM) at pH 5.5 under constant stirring. The liposomal solution was extruded five times through a polycarbonate membrane with a drain disk. To obtain a homogeneous population of liposomes, membranes with different pore size were used, 200 nm, 100 nm and 80 nm.
Ammonium sulphate buffer was exchanged with Hepes saline buffer (Hepes 10 mM, EDTA 5 mM and NaCl 150 mM; pH 6.7) through a PD-10 column and LPs were incubated with Dox at a molar ratio of 0.1:1(Dox:Lipid) in a thermoshaker (Vortemp 56, Labnet, USA) for 1 h at 60 °C. Non-encapsulated Dox was removed by ultracentrifugation at 40,000 rpm for 2 h at 4 °C and the final Dox liposomal formulation was kept in Hepes saline at 4 °C until use.
5
Quantifying Bacterial Gene Expression
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Plasmids containing the ompF-sfgfp reporter, pLux-MicF, or controls were transformed into E. coli MG1655, plated onto LB + Agar plates containing 34 μg/mL chloramphenicol and 100 μg/mL carbenicillin, and incubated overnight at 37°C. Three colonies from each condition were inoculated into 300 μL of LB with corresponding antibiotics in a 2 mL 96-well block (Costar 3961) sealed with a breath-easier membrane and grown for 17 hours overnight at 37°C while shaking at 100 rpm (Labnet Vortemp 56). Four microliters of the overnight culture were added to 296 μL of MOPS media that was pre-warmed at 37°C for 30 minutes. The cultures were grown under the same conditions as above for approximately 2.5–3 hours until an OD600 of 0.35–0.4 was reached. Cultures were diluted a second time using pre-warmed MOPS into a 96-well plate (Corning 3631) to reach an OD600 of approximately 0.015. AHL was added to each well at a final concentration of 2 nM. CPP-PNAs were added to each well at final concentrations indicated in the figures. The plate was sealed with a breathe-easy membrane and placed on a Biotek Synergy H1 plate reader. The temperature was controlled at 37°C with continuous double-orbital shaking (425 cpm) for two hours. OD600 and SFGFP fluorescence was measured from the bottom of the plate every ten minutes (485 nm excitation, 520 nm emission, gain 80).
6
Automated DNA Extraction from DBS Samples
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The DBS punching was performed using a Wallac DBS Puncher (PerkinElmer, Waltham, MA, USA) into wells of 96-well plates. The 3.2 mm diameter punches underwent semi-automated DNA extraction in a Tecan Robotic Liquid Handler (Tecan, US, Morrisville, NC, USA) using Generation DNA Purification and Elution Solutions (QIAGEN, Germantown, MD, USA). The extraction protocol included two purification washes of the DBS by adding 100 µL of Generation DNA Purification solution per well, followed by one wash with Generation DNA Elution Solution. All washes were performed at 37 °C for 10 min on a microplate shaker. After discarding the Elution buffer, 75 µL of new Elution solution was added to each well and incubated for 30 min at 98 °C while being shaken in a VorTemp 56 (Labnet, Edison, NJ, USA) at 1300 rpm. Plates were then cooled down to ambient temperature.
7
Rapid Diagnosis and Identification of Plasmodium Infections
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Plasmodium falciparum (Pf)/Pan-LDH rapid diagnostic test (RDT) was the product of Guangzhou Wondfo Biotechnology Co., Ltd., China (lot W05460602WC). CLIP-PCR 2.0 kits were purchased from Diacurate Technology Co., China (Daanrui@126.com), and multi-section CLIP-PCR (mCLIP-PCR) kits for Plasmodium genus and species identification were donated by Peking Union Medical College, Beijing. The related probe sequences are provided in Additional file 1: Table S1. An Omron non-contact infrared forehead thermometer (model MC-720) was the product of Kunshan Reying Photoelectric Co., Ltd. (China), a real-time fluorescence quantitative PCR (qPCR) instrument (model CG-5) was purchased from Shanghai Likang Biomedical Technology Holding Co., Ltd. (China), and a shaking incubator (VorTemp 56) was obtained from Labnet International (USA).
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Plasmodium falciparum (Pf)/Pan-LDH rapid diagnostic test (RDT) was the product of Guangzhou Wondfo Biotechnology Co., Ltd., China (lot W05460602WC). CLIP-PCR 2.0 kits were purchased from Diacurate Technology Co., China (Daanrui@126.com), and multi-section CLIP-PCR (mCLIP-PCR) kits for Plasmodium genus and species identification were donated by Peking Union Medical College, Beijing. The related probe sequences are provided in Additional file 1: Table S1. An Omron non-contact infrared forehead thermometer (model MC-720) was the product of Kunshan Reying Photoelectric Co., Ltd. (China), a real-time fluorescence quantitative PCR (qPCR) instrument (model CG-5) was purchased from Shanghai Likang Biomedical Technology Holding Co., Ltd. (China), and a shaking incubator (VorTemp 56) was obtained from Labnet International (USA).
8
Antisense RNA Expression in E. coli
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Each sense and antisense plasmid was transformed separately, or in combination, into chemically competent E. coli TG1 cells. Where indicated, a control antisense plasmid, lacking the antisense RNA sequence but containing a promoter and terminator (Supplementary Figure S1), was used to maintain a consistent cellular load to properly compare fluorescence levels with or without the antisense RNA present. Transformed cells were plated on LB+Agar media with 100 μg/ml carbenicillin, 34 μg/ml chloramphenicol or both and incubated overnight at 37°C. The next day, individual colonies were picked and grown in 1 ml of the appropriate LB+antibiotic in a 2 ml 96-well block (Costar) and grown approximately 17 h overnight at 37°C at 1000 rpm in a VorTemp 56 (Labnet) benchtop shaker. Twenty-four microliters of this overnight culture was then used to subculture into 1.2 ml of LB+antibiotic. E. coli TG1 cells without plasmids were prepared in the same way without antibiotic for probing endogenously expressed RNAs. The subculture was grown under the same conditions as the overnight culture for at least 3 h before measuring fluorescence (for cultures containing the sense plasmid with superfolder GFP; SFGFP) and performing structure probing. See Supplementary Methods (steps 17–21) for more details.
9
Determination of E. coli Doubling Time
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E. coli strains were isolated on LB-agar plates containing appropriate antibiotics if necessary. Six individual colonies from each condition were inoculated into 300 μl of LB with corresponding antibiotics, if necessary, in a 2 ml 96-well block (Costar 3961) sealed with a breath-easier membrane and grown for 17 hours overnight at 37°C while shaking at 100 rpm (Labnet Vortemp 56). Four microliters of the overnight culture were added to 296 μl of LB and grown under the same conditions for one hour. From that point, 2 μl from each culture was removed every 20 minutes and used to determine CFU/ml via dilutions and plating. The doubling time for an individual colony was determined by taking the log base 2 of each CFU/ml value, then determining the slope log2(CFU/ml) vs time and taking the inverse of that slope value. Doubling times were averaged across the six colonies.
10
Fluorescence-based Protein Expression Assay
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Plasmids were transformed into chemically competent E. coli strains, plated on LB-agar plates containing chloramphenicol and carbenicillin, and incubated overnight at 37°C. Three colonies from each condition were inoculated into 300 μl of LB with corresponding antibiotics in a 2 ml 96-well block (Costar 3961) sealed with a breath-easier membrane and grown for 17 hours overnight at 37°C while shaking at 100 rpm (Labnet Vortemp 56). Four microliters of the overnight culture were added to 296 μl of MOPS media that was pre-warmed at 37°C for 30 minutes. The cultures were grown under the same conditions as above for 2.5 or 3 hours depending on the strain used. One hundred microliters of each culture was transferred to a 96-well plate (Costar 3631), and OD600 and bulk sfGFP fluorescence (485 nm excitation, 520 nm emission) were measured using a Biotek Synergy H1 plate reader. Each experiment included two sets of controls: a media blank and E. coli transformed with control plasmids. OD and FL for each culture were first corrected by subtracting the mean value of the media blank. The ratio of the corrected FL and OD (FL/OD) was calculated for each culture. The E. coli cultures transformed with control plasmids were used to correct for autofluorescence. The average FL/OD from the control cultures was subtracted from FL/OD from each condition.
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