The culture conditions of the bacterial strains (A40-3, A40-4, B30-1, and B31-7) were the same as those described for A40-3 in “Transcriptomic analysis.” Bacterial cells were sampled at the initial phase (0 h) and the early and middle logarithmic growth phases in each of the three replicates. Total RNA was extracted using the RNeasy minikit (Qiagen, USA). Then, the RNA quality was determined as described in “Transcriptomic analysis.” The extracted RNA (200 ng) was subsequently reverse-transcribed using the TransScript all-in-one first-strand cDNA synthesis super mix for qPCR (Trans, China), and then qPCR was performed using a LightCycler II 480 system (Roche, Switzerland) following the instructions for SYBR Premix Ex Taq (TaKaRa, Japan). The cycling threshold (CT) values were calculated by using the LightCycler 480 software. The relative expression level was indicated as fold change, which was calculated using the LightCycler 480 software according to the 2−ΔΔCT method using the gene recA and the 0-h sample for normalization (55 (link)). The recA gene was used as the reference gene. The primers used in this experiment are listed in Table S4.
Light cycler 2 480 system
Manufactured by Roche
Sourced in Switzerland
The Light Cycler II 480 System is a real-time PCR instrument designed for precise quantification of nucleic acid targets. It features a high-performance optical system and advanced thermal control for reliable and sensitive detection of gene expression levels and DNA/RNA quantification.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Sybr premix ex taqtm, by Takara Bio (3 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Transscript all in one first strand cdna synthesis supermix for qpcr, by Transonic Systems (2 mentions)
Goldenstar rt6 cdna synthesis kit, by Tsingke (2 mentions)
2216e medium, by BD (1 mentions)
Sea salt, by Merck Group (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
5 protocols using light cycler 2 480 system
1
Quantitative Gene Expression Analysis
2
DMSP Regulation of Gene Expression
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The cells of ITI_1157 and ISM were cultured in 2216E medium (BD, America) at 40°C and 30°C respectively. When the experimental groups containing 2 mM DMSP and the control groups containing no DMSP grew to an OD600 of 0.8, RNA was extracted using the RNeasy mini kit (Qiagen, America), and was subsequently reverse‐transcribed to cDNA using Goldenstar™RT6 cDNA Synthesis Kit (TsingKe, China), and then qPCR was performed using a Light Cycler II 480 System (Roche, Switzerland) following the instructions of SYBR® Premix Ex TaqTM (TaKaRa, Japan) with the following cycling conditions: 95°C for 5 min, 45 cycles of 95°C for 10 s and 60°C for 30 s. The recA gene was used as the house keeping gene.
3
Transcriptional Response to Diaminoacid Exposure
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Bacteria were cultured in 2216E medium at 25°C to an OD600 of 0.8. The cells in the culture were collected by centrifugation at 4000 × g for 10 min at 4°C, washed three times with 3% sea salt solution, and then inoculated into the medium containing a single DAA, 50 mM glucose, 3% sea salt (Sigma, United States) and 0.2 M phosphate buffer (pH 8.0). Bacteria were collected at 0 h, and during early and middle logarithmic growth phase. Total RNA was extracted using the RNeasy® Mini Kit (QIAGEN, United States). The extracted RNA was subsequently reverse-transcribed using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Trans, China) and then qPCR was performed using a Light Cycler II 480 System (Roche, Switzerland) following the instructions of SYBR® Premix Ex TaqTM (TAKARA, Japan). The recA gene was used as the reference gene.
4
Transcriptomic response of Strain HTCC1062 to TMAO
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Strain HTCC1062 was firstly cultured in AMS1 medium amended with 25 μM glycine, 10 μM methionine and 50 μM pyruvate. When the concentration of cells reached 2 × 107 cells/ml, TMAO was added into the medium with a final concentration of 0.8 mM. The group without the addition of TMAO was set up as a control. After 0.5 or 2 h incubation, RNA was extracted from the cells using the RNeasy mini kit (Qiagen, America), and was subsequently reverse-transcribed to cDNA using Goldenstar™RT6 cDNA Synthesis Kit (TsingKe, China). The qPCR experiments were performed using a Light Cycler II 480 System (Roche, Switzerland) following the instructions of SYBR® Premix Ex TaqTM (TaKaRa, Japan) with the following cycling conditions: 95°C for 5 min, 45 cycles of 95°C for 10 s and 60°C for 30 s. The recA gene was used as an internal reference gene.
5
DMSP Induction of Psychrobacter sp. D2
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Cells of Psychrobacter sp. D2 were cultured in the marine broth 2,216 medium at 180 rpm at 15°C to an OD600 of 0.8. Then, cells were induced by 5 mM DMSP, and the control group without DMSP was also set up. After 20 min’s induction, total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Genomic DNA was removed using gDNA Eraser (TaKaRa, Japan) and cDNA was synthesized using a PrimeScript RT reagent Kit. The qPCR was performed on the Light Cycler II 480 System (Roche, Switzerland) using a SYBR Premix Ex Taq (TaKaRa, Japan). Relative expression levels of target genes were calculated using the LightCycler480 software with the ‘Advanced Relative Quantification’ method. The recA gene was used as an internal reference gene. The primers used in this study are shown in Supplementary file 1h .
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