DNA extraction was performed using 250 mg of soil with E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following manufacturer's instructions. Quantification and quality check of the extracts were performed using a Quawell Q3000 microvolume spectrophotometer (Quawell Technology, San Jose, USA). Library preparation was performed using the 16S Barcoding Kit 1-24 (Oxford Nanopore Technologies, Oxford, UK) following manufacturer's instructions. The full-length 16S rRNA bacterial gene was amplified through PCR using the kit's barcoded primers (27F: 5′-AGA GTT TGATCMTGG CTC AG-3′ and 1492R: 5′-CGG TTA CCT TGT TAC GAC TT-3′) allowing multiplexing. Amplification was conducted using repliQa HiFi ToughMix (Quantabio, Beverly, MA, USA) in Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) at the following conditions: initial denaturation at 95 °C for 1 min followed by 25 cycles of denaturation (95 °C, 20 s), annealing (55 °C, 30 s), and extension (65 °C, 2 min), with a final extension at 65 °C for 5 min. After amplification followed the PCR product purification with Agencourt AMPure XP beads (Beckman Coulter, CA, USA). The concentration of purified DNA amplicons was determined using the microvolume spectrophotometer.
Q3000 microvolume spectrophotometer
Manufactured by Quawell
Sourced in United States
The Q3000 microvolume spectrophotometer is a compact and accurate instrument designed for the quantification of nucleic acids and proteins in small sample volumes. The core function of the Q3000 is to measure the absorbance of light by a sample, which can be used to determine the concentration of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr master mix, by Thermo Fisher Scientific (3 mentions)
Maxima first strand cdna synthesis kit with thermolabile dsdnase, by Thermo Fisher Scientific (3 mentions)
Quick rna miniprep kit, by Zymo Research (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
16s barcoding kit 1 24, by Oxford Nanopore (1 mentions)
Repliqa hifi toughmix, by Quantabio (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
E z n a soil dna kit, by Omega Bio-Tek (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Amplitaq gold 360 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Multicolor real time pcr detection system, by Bio-Rad (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
5 protocols using q3000 microvolume spectrophotometer
1
Soil Microbiome 16S rRNA Sequencing
2
Gene Expression Analysis by qPCR
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Whole RNA was isolated by a Quick RNA Miniprep kit (ZYMO Research) and quantified by Q3000 micro volume spectrophotometer (Quawell). RNAs were retrotranscribed on a Maxima First Strand cDNA Synthesis Kit with Thermolabile dsDNase (ThermoFisher). Real-time qPCR was carried out using the PowerUp SYBR Master Mix (Thermofisher) and 7500 Fast Real-Time PCR System (ThermoFisher).
Gene expression was quantified by the ΔΔCt method 15 . Sample values were normalized to the threshold value of housekeeping gene GAPDH.
Chromatin immunoprecipitated fragments were amplified by AmpliTaq Gold 360 DNA Polymerase (ThermoFisher) using PTC-1148 MJ Mini Thermal Cycler (Bio-Rad).
The primers used for amplification are indicated in Tables S3 andS4.
Gene expression was quantified by the ΔΔCt method 15 . Sample values were normalized to the threshold value of housekeeping gene GAPDH.
Chromatin immunoprecipitated fragments were amplified by AmpliTaq Gold 360 DNA Polymerase (ThermoFisher) using PTC-1148 MJ Mini Thermal Cycler (Bio-Rad).
The primers used for amplification are indicated in Tables S3 andS4.
3
Quantifying GAP43 Gene Expression
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Whole RNA was isolated by a Quick RNA Miniprep kit (ZYMO Research) and quantified by a Q3000 microvolume spectrophotometer (Quawell, San Jose, CA, USA). RNAs were retrotranscribed on a Maxima First Strand cDNA Synthesis Kit with Thermolabile dsDNase (Thermo Fisher Scientific, Madrid, Spain). Real-time quantitative polymerase chain reaction (qPCR) was carried out using the PowerUp SYBR Master Mix (Thermo fisher Scientific, Madrid, Spain) and StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Madrid, Spain). Gene expression of GAP43 was quantified by the ΔΔCt method. Sample values were normalized to the threshold value of the housekeeping gene GAPDH.
4
Quantitative Gene Expression Analysis
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Total RNA was extracted from ESCs using Quick RNA Miniprep kit (ZYMO Research); quantity and integrity were measured on a Q3000 micro volume spectrophotometer (Quawell). RNAs were reverse transcribed by a Maxima First Strand cDNA Synthesis Kit with Thermolabile dsDNase (Thermo Fisher Scientific). Real-time qPCR was carried out on a PowerUp SYBR Master Mix (Thermo Fisher Scientific) and ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The reactions were run four times (independent biological experiments). The primers used for amplification are indicated in Supplementary Table 3 . The fractional cycle number at which fluorescence passed the threshold (Ct values) was used for gene expression quantification by the comparative ΔΔCt method. Sample values were normalized to the threshold value of Gapdh housekeeping gene.
5
Hippocampal TPH2 and IDO1 mRNA Expression
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The levels of TPH2 and IDO1 mRNAs in the hippocampus were evaluated by qRT-PCR assay. The total RNA of hypothalamus was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Then, the concentration and quality of total RNA were determined by Q3000 micro-volume spectrophotometer (Quawell Technology, San Jose, CA, USA) and 1% agarose gel electrophoresis. The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used to synthesize first-strand cDNA on C1000 Touch TM Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the standard protocol. The SYBR® Green PCR Master Mix (Thermo Fisher Scientific) was used to amplify the cDNA in the Multicolor Real-time PCR Detection System (Bio-Rad Laboratories Inc.), and the cycling parameters were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 56°C (TPH2) or 58°C (IDO1) for 1 minute, then followed by 65°C for 5 seconds and 95°C for 15 seconds. The 2−∆∆Ct method was used to calculate the expression of mRNA. Table 1 shows the sequences for primers, which were designed according to the published mRNA sequences in NCBI, and GAPDH (BBI Life Science, Amherst, MA, USA) was used as the house-keeping gene in the experiment.
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