An African-American family segregating autosomal dominant POAG was ascertained through ophthalmic records in the Department of Ophthalmology and Visual Sciences at Washington University School of Medicine. Blood samples were obtained from available family members including five affected males, one unaffected male, two unaffected females, and ten individuals in the third generation of unknown disease status (Fig 1 ). Leukocyte genomic DNA was purified using the Gentra Puregene Blood kit (Qiagen, Valencia, CA), and quantified by absorbance at 260 nm (NanoDrop 2000, Wilmington, DE).
Nanodrop 2000
Manufactured by Qiagen
Sourced in United States, Germany
The NanoDrop 2000 is a spectrophotometer designed for the quantification and analysis of nucleic acids and proteins. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to measure the absorbance across a wide range of wavelengths. The NanoDrop 2000 provides accurate and reproducible results for a variety of applications, including DNA, RNA, and protein concentration determination.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Gentra puregene blood kit, by Qiagen (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Sybr green dye, by Thermo Fisher Scientific (2 mentions)
7500 system sds software version 1, by Thermo Fisher Scientific (2 mentions)
Minelute kit, by Qiagen (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
On column dnase 1 digestion, by Qiagen (1 mentions)
Rneasy rna extraction kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Novaseq pe250 platform, by Illumina (1 mentions)
36 protocols using nanodrop 2000
1
Genetic Investigation of Autosomal Dominant POAG
2
Fecal Microbiome Analysis Post-FMT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fecal specimens were collected 2 weeks after FMT in sterile 2-mL tubes containing pure chilled ethanol, frozen within 30 min, and stored at − 80°C until analysis. Genomic DNA was extracted using the cetyltrimethylammonium bromide method. An equivalent of 1 μL of each sample was used for DNA quantification using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The amplification of the V3-V4 region of the 16S rRNA was performed to analyze the bacterial population and execute amplification of the variable region. Polymerase chain reaction (PCR) was conducted using the bacterial universal forward primers 319F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and the reverse 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). The PCR products were verified by electrophoresis on 1% (w/v) agarose gels in Tris-borate-EDTA (TBE) buffer stained with Genecolour I (GeneBio Systems, Oakville, ON, Canada) and visualized under ultraviolet (UV) light. Amplicons were first purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), quantified using NanoDrop 2000, and then pooled in equal concentrations. Pooled amplicons (2 nmol/ L) were subjected to sequencing using an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA), following the standard Illumina platform protocols.
3
Gene Expression Analysis by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was purified from cells and tissues using RNeasy RNA extraction kit (Qiagen) as per the manufacturer's instructions. Genomic DNA was eliminated using on-column DNase I digestion (Qiagen) and NanoDrop 2000 was used in order to measure RNA quality. cDNA was prepared from equal amounts of total RNA (50–1,000 ng), using iScript cDNA synthesis kit (Bio-Rad). The following primers were used—Gapdh: 5′-GCTGACCTGCTGGATTACATT-3′ (forward), 5′-GTTGAGAGATCATCTCCACCA-3′ (reverse); Tnfa: 5′-CCCAGGCAGTCAGATCATCTTC-3′ (forward), 5′-GCTTGAGGGTTTGCTACAACATG-3′ (reverse); iNos: 5′-CGCCTTCAACACCAAGGTTG-3′ (forward), 5′TGGGGACAGTCTCCATTCCCA-3′; Il6: 5′-TCAGGAAATTTGCCTATTGAAAATTT-3′ (forward), 5′-GCTTTGTCTTTCTTGTTATCTTTTAAGTTGT-3′ (reverse); Ccl5: 5′-GAGTGACAAACACGACTGCAAGAT-3′ (forward), 5′-CTGCTTTGCCTACCTCTCCC T-3′ (reverse); Kc: 5′-CCATGGCTGGGATTCACC-3′ (forward), 5′GACCATTCTTGAGTGTGGCTATGAC-3′ (reverse); 16S V6: 5′-TCGATGCAACGCGAAGAA-3′-(forward), 5′-ACATTTCACAACACGAGCTGACGA-3′ (reverse). Gene expression levels were normalized to Gapdh as housekeeping gene. Relative mRNA expressions were calculated according to the 2−ΔΔt calculation method. Specificity of PCR amplifications was assessed by melting curve analysis.
4
Quantitative RT-PCR Analysis of Gene Expression
5
Amplification and Sequencing of Microbial Genomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total genomic DNA from each of the 24 samples was extracted using a FastDNA® Spin Kit (MP Biomedicals, Santa Ana, CA, United States) based on the manufacturer’s instruction. The purity and concentration of genomic DNA were assessed using agarose gel electrophoresis and the NanoDrop-2000 (Thermo Fisher Scientific, Waltham, MA, United States). The DNA concentration was diluted to 30 ng/μl for each sample. For PCR amplification, the bacterial hypervariable V4 region of the 16S rRNA gene was amplified using primers 515F/806R (Moreau and Rubin, 2017 (link)), and the fungal ITS1 region was amplified using primers ITS1/ITS2 (Zhang et al., 2010 (link)). The PCR reaction solutions and thermocycling steps for V4 and ITS1 regions followed Wei et al. (2018) (link). The PCR products were visualized on 2% agarose gel electrophoresis, quantified by NanoDrop-2000, and purified with the Qiagen Gel Extraction Kit (Qiagen, Dusseldorf, Germany). Libraries were generated for each sample as described in Xiang et al. (2022) (link). Finally, the libraries were sequenced on an Illumina NovaSeq PE250 platform at Novogene Bioinformatics Technology Co., Tianjin, China, generating 250 bp paired-end reads.
6
Autosomal Dominant Cataract Pedigrees
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three Caucasian-American pedigrees segregating autosomal dominant cataract were ascertained through ophthalmic records in the Department of Ophthalmology and Visual Sciences at Washington University School of Medicine. Blood samples were obtained from available family members including a spouse (Figures 1 and 2 ). Leukocyte genomic DNA was purified using the Gentra Puregene Blood kit (Qiagen, Valencia, CA) and quantified by absorbance at 260 nm (NanoDrop 2000, Wilmington, DE).
7
PCR Amplification and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Residual primers and nucleotides were purified from PCR amplifications using QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA, USA) and DNA concentration was measured using spectrophotometer (NanoDrop2000). Equimolar amounts of the 22 PCR amplified products were pooled into a tube as one sample to ensure equivalent hybridization; and the sample was further processed using the GeneChip Resequencing Assay Kit.
8
Bacterial 16S rDNA Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from strain RM, using QIAGEN (CA, USA) DNA extraction kit, and its concentration was determined using a UV–Vis spectrophotometer (NanoDrop 2000). Fragments of 16S rDNA were amplified using universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-CCCCGTCAATTCATTTGAGTTT-3′). PCR product was purified using a QIAGEN PCR purification kit and sequenced using an automated ABI PRISM 3700 sequencer (USA). The obtained 16S rRNA sequence was compared against the available sequence database using BLAST program in NCBI website. Phylogenetic tree was constructed using neighbour-joining distance method by software Mega 6.0.
9
Rabbit Microarray Transcriptome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The eight samples from each group were used as biological replicates for the microarray analysis. RNA from each placental tissue (200 ng) was amplified and labelled with cyanine 3 (Cy3) dye using the One-colour Microarray-based Gene Expression Analysis Low Input Quick Amp Labelling Kit (Agilent Technologies). Excess dye was removed with the RNeasy Mini Kit (Qiagen) and dye incorporation and concentration were determined using the microarray setting on the Nanodrop 2000 and the Bioanalyzer 2100. Hybridization was performed at the CRB GADIE (INRA Jouy-en-Josas, France, http://crb-gadie.inra.fr/ ). Equal amounts of Cy3-labelled samples (600 ng, Specific Activity > 6.0 pmol Cy3/μg of cRNA) were mixed with blocking agent and fragmentation buffer, and then 24 μl of the mixture was hybridized into the Rabbit Custom Gene Expression Microarrays (GEO accession GPL18913, Agilent-042421 Rabbit BDR version 2, Agilent Technologies) for transcriptomic analysis (12775 genes)66 (link). After 17 h at 65 °C, the hybridized slides were washed twice for 1 min at room temperature in GE Wash Buffer 1 and 1 min at 37 °C with GE Wash Buffer 2 (Agilent Technologies) and air-dried and scanned immediately.
10
SYBR-Green Quantitative Real-Time PCR for MeDIP Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microarray data at the Pparg locus were verified by SYBR-green based real-time PCR analysis of the adaptor-mediated PCR products from the MeDIP samples (LG-SF and LG-SC groups). PCR products were purified using the MinElute kit (Qiagen) and quantified using the Nanodrop 2000. Twenty nanograms of purified amplicon were subjected to real-time PCR. The reaction consisted of 1× ABI master mix containing Taq polymerase, dNTPs, SYBR green dye and ROX as passive dye (Life Technologies, Carlsbad, CA, USA) and 200 nM of specific primers (Supplementary Material Table S1 ). The PCR program started with a Taq polymerase activation step (10 min at 95°C) followed by 40 cycles at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. Data analyses were performed using the 7500 System SDS software version 1.4 (Applied Biosystems). A fragment within the Actb locus was used as calibrator. Adaptor-mediated PCR products from the input fractions were used as reference to calculate the IP/INPUT enrichment for the target (IP/IN target) and the calibrator (IP/IN calibrator) loci. Fold change enrichment (FCE) was calculated using the equation: FCE=2ˆ-(IP/IN target- IP/IN calibrator).
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!