Total RNA was extracted from exponentially growing bacterial cultures grown with or without specific stress as described above. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600=0.4–0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by resuspending in 1 ml TRIzol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 s. The procedure was repeated for 2–3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13,000 rpm for 10 min; supernatant was collected and processed for RNA isolation using Direct-Zol RNA isolation kit (ZYMO). Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluorometer (Invitrogen).
Nanodrop nd 1000 spectrophotometer
Manufactured by Promega
Sourced in Germany, Italy, United States
The NanoDrop ND-1000 spectrophotometer is a laboratory instrument used to measure the concentration and purity of nucleic acid and protein samples. It utilizes a patented sample retention technology that requires only 1-2 microliters of sample for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Fastprep 24 bead beater, by MP Biomedicals (2 mentions)
Dnase 1, by Promega (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Direct zol rna isolation kit, by Zymo Research (2 mentions)
Lysing matrix b, by MP Biomedicals (2 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Ribo zero plant kit, by Illumina (1 mentions)
Clc genomics workbench 12, by Qiagen (1 mentions)
Nucleospin rna clean up xs, by Macherey-Nagel (1 mentions)
3500 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Pop7 polymer, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector system kit, by Promega (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Plasmid miniprep kit, by Promega (1 mentions)
Taqman probe, by Roche (1 mentions)
Quantifluor st fluorometer, by Promega (1 mentions)
454 life sciences genome sequencer flx system, by Roche (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
5 protocols using nanodrop nd 1000 spectrophotometer
1
Total RNA Extraction from Bacterial Cultures
2
Bacterial Total RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from exponentially growing bacterial cultures grown with or without specific stress as described above. Briefly, 25 ml of bacterial culture was grown to the mid-log phase (OD600=0.4–0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween-80. Cells were pelleted by centrifugation and lysed by resuspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 s. The procedure was repeated for 2–3 cycles with incubation on ice in between pulses. Next, CLs were centrifuged at 13,000 rpm for 10 min; the supernatant was collected and processed for RNA isolation using Direct-Zol RNA isolation kit (ZYMO). Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluorometer (Invitrogen).
3
Total RNA Extraction and RNA-seq Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 100 mg of fresh leaf material using the Spectrum Total Plant RNA Kit (Sigma Aldrich N.V). Quantification and quality controls were done with Nanodrop ND-1000 spectrophotometer and Quantus (QuantiFluor® RNA System kit (Promega Benelux B.V.) followed by RNA-purification (NucleoSpin® RNA Clean-up XS; Machery-Nagel, Germany). Library preparation and rRNA-depletion were done externally (Admera Health, South Plainfield, NJ, USA) using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® and Ribozero Plant kit, respectively, followed by NextSeq sequencing (2 × 150 bp read length, 2 × 20 M reads per sample). The obtained sequence reads were subjected to quality filtering, adapter removal and a standardized bioinformatics analysis strategy using Cutadapt, Pear, SortmeRNA and the VirusDetect pipeline [40 (link)]. The consensus sequences of the complete genomes were obtained through reference mapping in CLC Genomics Workbench 12 (Qiagen, Vedbæk, Denmark).
4
Cloning and Sequencing of Bacterial 16S rDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified PCR products were sequenced and cloned into the PGEM vector following the instructions provided by the pGEM®-T Easy Vector System kit (Promega, Italy) using Escherichia coli competent cells as a host. The obtained plasmids, pGEM-BB (pGEM + B. breve) and pGEM-LS (pGEM + L. salivarius), were extracted (Plasmid Miniprep Kit, Promega, Italy), quantified using the NanoDrop ND-1000 spectrophotometer and diluted. The dilutions, which ranged from 106 to 101 vector copy numbers, were used as standards in the quantitative RT-PCR (qRT-PCR) assays. The cloned fragments of 16S rDNA in the pGEM-BB and pGEM-LS vectors were amplified and sequenced with an automated sequence analyser (Genetic Analyser 3500, Applied Biosystems, CA, USA) using a 50-cm capillary array and a POP-7 polymer (Applied Biosystems) and the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, version 3.1) according to the manufacturer’s instructions. All electropherograms were manually edited for base ambiguity. The obtained FASTA sequences were aligned using CLUSTAL-W software (http://www.ebi.ac.uk/clustalw/ ) and used for the design of species-specific primers and TaqMan probes (Roche Diagnostics, Mannheim, Germany) (Supplementary Table 1 ).
5
16S rRNA Gene Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bacterial genomic DNA was amplified with the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene33 (link)34 (link)45 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode at the 5′ end of the reverse primer to allow all samples to be included in the single 454 FLX sequencing run. All PCR reactions were performed in 50-μl triplicates and combined after PCR. The products were extracted with the QIAquick Gel Extraction Kit (QIAGEN) and quantified on NanoDrop ND-1000 spectrophotometer, QuantiFluor-ST Fluorometer (Promega, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Equimolar concentrations of 83 samples were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!