The cultures were centrifuged at 8,000 × g to precipitate bacterial cells. Total RNA was extracted using the hot phenol method as previously described with modifications (11 (link)). Subsequently, the bacterial cells were washed two times with RNAse-free saline or phosphate-buffered saline (PBS; cat. no. E607016-0500; BBI solutions, Cardiff, UK). Then, 400–600 µl TES solution was added according to the precipitation amount, and the bacterial cells were resuspended. The same amount of phenol-water (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added followed by violent mixing. Centrifuge tubes containing a mixture of each sample, TES, and phenol-water were agitated at 65°C for 30–60 min in a Thermomixer Compact 5350 (Eppendorf, Hamburg, Germany), and then, the tubes were placed on ice and allowed to stand for 5 min. Then, the mixtures were centrifuged at 11,000 × g for 10 min at 4°C. The upper aqueous phase was selected and transferred to a new tube. Subsequently, a 1/2 volume of TRK-1002 lysis-solution and 2/3 volume of 95% ethyl alcohol was added to the upper aqueous phase, followed by vortex blending. Total RNA was then extracted using a TRK-1002 Purification kit (LC Sciences, Houston TX, USA), following the manufacturer's instructions. RNA quality was evaluated using an Agilent Bioanalyser (Agilent Technologies, Inc., Santa Clara, CA, USA).
Thermomixer compact 5350
Manufactured by Eppendorf
Sourced in Germany
The Thermomixer compact 5350 is a temperature-controlled mixing device for sample preparation in the laboratory. It provides precise temperature control and mixing of samples in microtubes, microplates, and other small vessels. The device offers a temperature range of 0°C to 99°C and a mixing speed of 300 to 3,000 rpm.
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Lab products found in correlation
3 protocols using thermomixer compact 5350
1
Total RNA Extraction from Bacterial Cells
2
Fecal Steroid Extraction and Quantification in Golden Hamsters
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Faecal steroids were extracted using the methanol-based procedure outlined in Palme et al. (2013 ). Lyophilisation was not necessary due to the consistency of golden hamster faeces. After homogenisation of each faecal sample (by using a mortar), 50 mg were weighed and stored at − 20 °C until assayed. Each faecal sample was shaken on a vortex (Velp Scientifica Vortex mixer, USA) at 20 rpm with 1 ml of 80% methanol and subsequently on a thermomixer (Eppendorf Thermomixer compact 5350, Eppendorf, Germany) at 500 rpm for 15 min. The suspension was then centrifuged at 2500×g for 5 min (Eppendorf centrifuge 5424, Eppendorf, Germany). Faecal cortisol metabolites were quantified in an aliquot of the extract (50 μl further diluted 1:10 with assay buffer) using a group specific 11-oxoaetiocholanolone enzyme immunoassay (EIA measuring glucocorticoid metabolites with a 5β-3α-ol-11-one structure) successfully validated for the golden hamster (Chelini et al. 2010 (link)). Details of this assay were first described by Möstl et al. (2002 (link)). Concentrations of FCM are expressed as nanograms per gram faecal dry matter.
3
Efficient Microbial DNA Extraction Optimized
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DNA extraction from the microorganisms collected by filtration (GF/B filters) was performed using silica membranes from a commercial Genomic Mini AX Bacteria + kit (A&A Biotechnology, Gdańsk, Poland), with some modifications to the manufacturer’s instruction, in order to lyse the microbial community more efficiently. Additionally, the tubes were continuously shaken (500 RPM, Eppendorf Thermomixer compact 5350) for 3 h. Centrifugation for 20 min at 50 °C and 650 RPM was added to the first step. Moreover, a thermal shock was also added to the fourth step of the manufacturer’s instructions, i.e., the tubes were placed five times at –20 °C for 2 min, then moved to +20 °C for 2 min and centrifuged each time. All laboratory procedures were conducted with sterile equipment, and all steps were carried out in a sterile laminar flow hood to avoid cross-contamination of the samples. Extracted DNA was quantified using a NanoDrop ND-1000 UV-vis (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C for further analyses.
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