Overnight cultures of S. aureus were diluted 1:100 in TSB and grown at 37°C. The cells were collected at the indicated cell density and processed with 1 ml RNAiso (TaKaRa) in combination with 0.1-mm-diameter zirconia-silica beads in a FastPrep-24 automated system (MP Biomedicals). The residual DNA was removed with RNase-free DNase I (TaKaRa). Transcription analysis of savRS was performed by reverse transcription-PCR using Moloney murine leukemia virus reverse transcriptase (TaKaRa) with primer RT-savS, and the PCR products were analyzed with the primers savS-F/savS-R and savS-F/savR-R. Reverse transcription for qRT-PCR analysis was performed using a PrimeScript first-strand cDNA synthesis kit (TaKaRa) with random primers. Real-time PCR was carried out with SYBR premix Ex Taq (TaKaRa) using a StepOne real-time PCR system (Applied Biosystems). The quantity of cDNA measured was normalized to the hu cDNA abundance. The primers used in this study are listed in Table 3 .
Fastprep 24 automated system
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep-24 is an automated system designed for efficient sample homogenization and cell lysis. It utilizes high-speed bead beating technology to disrupt a wide range of sample types, including tissues, cells, and microorganisms, in a reliable and reproducible manner.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq, by Takara Bio (6 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (5 mentions)
Rnaiso plus, by Takara Bio (5 mentions)
Rnase free dnase 1, by Takara Bio (3 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (3 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (2 mentions)
Rnaiso, by Takara Bio (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Takara Bio (1 mentions)
Recombinant dnase 1 rnase free, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Lc96 real time pcr system, by Roche (1 mentions)
Rnase free dnase 1, by Transgene (1 mentions)
Stepone real time system, by Thermo Fisher Scientific (1 mentions)
6 protocols using fastprep 24 automated system
1
Transcriptional Analysis of savRS in S. aureus
2
Bacterial RNA Extraction and qRT-PCR Analysis
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The twice-passaged overnight bacterial culture was transferred to blank TSB at an initial OD600 = 0.05, shaken at 37 °C until reaching OD600 = 2.5 in the middle stage of exponential growth, and then collected. The supernatant was discarded by centrifugation to collect the bacterial cells. To resuspend the cells, 900 μL of RNAiso Plus (TaKaRa, Japan) was added and then transferred to a crushing tube containing 0.6 g of zirconia-silica beads with a diameter of 0.1 mm. Further fragmentation was performed twice with a FastPrep-24 automated system (MP Biomedicals Solon, America) with an interval of 5 min. Residual DNA was removed with RNase-free recombinant DNase I (TaKaRa, Japan, 5 U/µL). For reverse transcription, using random primers, cDNA was synthesized with the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Quantitative reverse transcription-PCR (qRT-PCR) was performed using the StepOne Real-Time PCR System (Applied Biosystems, America) using SYBR Premix Ex Taq (TaKaRa, Japan). The amount of cDNA was measured by the 2−ΔΔCt method, using pta as the reference gene and the corresponding control samples as running calibrators. Table 3 lists the primers used in this study. All qRT-PCR assays were repeated at least three times.
3
Quantitative Transcriptional Analysis of S. aureus
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The overnight cultures of S. aureus were diluted to an OD600 of 0.05 in fresh TSB and cultivated to mid-exponential phase (OD600 = 1). The cells were collected by centrifugation at 12,000 × g for 1 min and then immediately treated with 0.9 mL of RNAiso plus (108-95-2, TaKaRa) and lysed with 0.1-mmdiameter-silica beads in a FastPrep-24 automated system (MP Biomedicals). The residual DNA removal and cDNA synthesis were achieved by using a PrimeScript RT reagent kit with gDNA eraser (RR047A, TaKaRa). qRT-PCR was performed with SYBR Ex Taq premix (RR420A, TaKaRa) using the StepOne real-time PCR system (Applied Biosystems). The cDNA quantity measured by real-time PCR was normalized to the abundance of pta cDNA63 (link). All qRT-PCR assays were repeated at least three times.
4
Quantitative Transcriptional Analysis of S. aureus
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Overnight cultures of S. aureus were diluted 1:100 in TSB and then grown to the indicated cell density until being collected. S. aureus cells were collected by centrifugation and processed with 1 ml of RNAiso plus (TaKaRa) in combination with 0.1-mm-diameter silica-zirconia beads in a FastPrep-24 automated system (MP Biomedicals), and the residual DNA was removed with RNase-free DNase I (TransGen, 3 U/μl). The concentration of total RNA was adjusted to 200 ng/μl. For the reverse transcription, the cDNAs were synthesized with a PrimeScript 1st Strand cDNA synthesis kit (TaKaRa) using random primers and real-time quantitative reverse transcription-PCR (qRT-PCR) was performed with SYBR Premix Ex Taq (TaKaRa) using the StepOne Real-Time PCR System (Applied Biosystems) and LC96 Real-Time PCR System (Roche). The quantity of cDNA measured by real-time PCR was normalized to the abundance of hu cDNA [49 (link)], and the corresponding control sample as the run calibrator. All qRT-PCR assays were repeated at least three times.
5
Antibiotic Stress Response in S. aureus
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Normally, the overnight cultures of S. aureus were diluted 1:100 in TSB with 15 μg/mL chloramphenicol, grown to the early exponential (OD600 = 0.5), and collected. When the stress response of S. aureus to antibiotics was detected, 10-fold MIC level were added at the early exponential (OD600 = 0.5) and then cultured for 30 min. The collected cells were processed with 1 mL of RNAiso Plus (TaKaRa) in combination with 0.1-mm-diameter-silica beads in a FastPrep-24 automated system (MP biomedicals Solon, OH, United States), and then used RNase-free DNase I (TaKaRa) to remove the residual DNA. The concentration of total RNA was adjusted to 200 ng/μL. Reverse transcription was carried out with the PrimeScript 1st Strand cDNA synthesis kit (Takara) and real-time quantitative reverse transcription-PCR (RT-qPCR) was performed with SYBR Premix Ex Taq (TaKaRa) using a StepOne realtime system (Applied Biosystems). The relative quantity of cDNA measured by real-time PCR was normalized to the average abundance of wild-type strain samples using housekeeping gene hu as the reference gene (Valihrach and Demnerova, 2012 (link)).
6
Quantifying S. aureus gene expression
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The S. aureus cells were collected at the indicated time points to isolate the total RNA. Then, the cells were treated with 1 ml of RNAiso Plus (TaKaRa, Kyoto, Japan) and 0.1mm diameter silica beads in a FastPrep-24 Automated system (MP Biomedicals, Solon, OH, USA), and the residual DNA was removed using RNase-free DNase I (TaKaRa, Kyoto, Japan). For reverse-transcription, the cDNAs were synthesized using the PrimeScript first-strand cDNA synthesis kit (TaKaRa, Kyoto, Japan). Real-time PCR was performed using the SYBR Premix Ex Taq (TaKaRa, Kyoto, Japan) and the StepOne real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The quantity of the cDNA measured by real-time PCR was normalized to the quantity of the pta cDNA [18] (link). The results represent the means of three independent experiments.
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