Total genomic DNA was extracted from adult worms, using the commercial QiAamp DNA extraction kit (Qiagen). In the case of adult worms, only a single specimen was used in each DNA extraction. Briefly, the sample was enzymatically digested in 180 μL of a lysis solution (ATL buffer-Qiagen); then 20 μL proteinase-K (50 g/mL) was added and incubated at 56°C for 2-3 h with brief vortexing every 30 min. For eggs, the incubation time was set 1-2 h longer. The mixture, after adding 200 μL buffer AL (Qiagen) containing guanidine hydrochloride and 4 μL RNase A (100 mg/mL) and mixing by pulse-vortexing for 15 s, was further incubated at 70°C for 10 min. Thereafter, 200 μL of ethanol (96–100%) was added and mixed by vortexing for 15–20 s. The contents were then loaded onto a QIAamp Spin Column for DNA binding and spin down for 1 min. The column with the DNA bound was washed several times using solutions (AW1; AW2 buffers—Qiagen) provided according to manufacturer's instruction. Finally, the genomic DNA was eluted in 50 μL elution buffer (AE) and stored at −20°C until use. The DNA from fecal samples was extracted using QIAamp DNA Stool Mini Kit according to the manufacturer's instructions, and the DNA from metacercariae in fish tissue was extracted according to a previous description [9 (link), 13 (link)]. The concentration of DNA samples was estimated using Thermo Nanodrop 1000.
Thermo nanodrop 1000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo NanoDrop 1000 is a UV-Vis spectrophotometer designed for the measurement of small sample volumes. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform absorbance measurements in the 220-750 nm wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Atl buffer, by Qiagen (1 mentions)
Buffer al, by Qiagen (1 mentions)
Qiaamp dna extraction kit, by Qiagen (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Dnaman 8, by Lynnon Biosoft (1 mentions)
Pcr machine, by Thermo Fisher Scientific (1 mentions)
Qiasymphony sp, by Qiagen (1 mentions)
Ex taq hot start version kit, by Takara Bio (1 mentions)
Abi 3730xl sequencing machine, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Chemiscope 3400 mini, by Clinx (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Ncode mirna first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using thermo nanodrop 1000
1
Genomic DNA Extraction from Parasitic Worms
2
Potato leaf RNA extraction and cDNA analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from potato leaves using TRIzol™ Reagent (Invitrogen Co., United Kingdom) according to the manufacturer’s instructions. RNA concentrations were measured using a Thermo NanoDrop 1000 spectrophotometer system (Thermo Fisher Scientific Co., United States). cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega Co., United States), and then was used as a template for subsequent PCR amplifications. Based on transcript sequences, cDNA sequences encoding ORFs of StCBL4 and StCIPK2 were amplified with primers designed using CE Design V1.04 (Supplementary Table 2 ) according to amino acid sequence alignments conducted using SMART.1 Next, multiple sequence alignments were performed using DNAMAN 8.0 software (Lynnon Biosoft Co., United States). Next, a phylogenetic tree was constructed based on alignments of amino acid sequences via the neighbor-joining (NJ) method using MEGA 5.0 software. Finally, bootstrap analysis with 1,000 replicates was performed to assess the statistical reliability of each branch of the tree.
3
Lung Cancer Specimen and Cell Line Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
Patient specimens and cell lines We randomly selected a cohort of ten lung cancer specimens from the Guangdong Lung Cancer Institute of Guangdong General Hospital in 2016. All samples, which were stored at −80°C after being frozen in liquid nitrogen, were assessed by two pathologists to ensure that more than 50% of the sample consisted of tumor tissue. We used nine nonsmall cell lung cancer cell lines (H460, PC9, H1650, H1975, A549, GLC82, L78, HCC827, and H2228), which were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai.
Reagents and instruments QIAsymphony DNA Mini Kit (Qiagen, Valencia, Germany); LungCarta ™ kit, PCR Accessory Set, iPLEX Pro Reagent Kit and SpectroCHIP® (Agena Bioscience, San Diego, CA, USA); H2O (Sigma-Aldrich, St. Louis, MO, USA); QIAsymphony SP (Qiagen, Valencia, Germany); Ex Taq ™ Hot Start Version Kit (Takara Biotechnology, Dalian, China); Thermo NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA); MassARRAY® Nanodispenser and MassARRAY® Analyzer (Agena Bioscience, San Diego, CA, USA); ABI 3730xl Sequencing Machine; and PCR Machine (Life Technologies, Carlsbad, CA, USA) were used.
+ Expand
4
Investigating STAT3/5 Signaling in RA Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells from RA patients and healthy controls, with or without stimulation by LPS, were collected and washed three times with sterile PBS. For total cellular protein, cells were lysed with RIPA Lysis Buffer (Beyotime, Nantong, China), supplemented with PMSF (Beyotime) and Protein phosphatase inhibitors (Boster, Wuhan, China). The proteins were quantitated using Thermo NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). Equal amounts of proteins (200 μg) were electrophoresed in 10% sodium dodecyl SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad) pre-soaked with 100% methanol. Non-specific binding was blocked for 1 hr at room temperature in Tris-buffered saline/0.05% Tween-20 (TBST) containing 5% non-fat milk powder, followed by incubation with the rabbit anti-human Ab against STAT3, STAT5, phospho-STAT3 (Tyr705, p-STAT3), phospho-STAT5 (Tyr694, p-STAT5) and GAPDH (Cell Signaling Technology Inc, Beverly, MA, USA) overnight at 4°C. Blots were washed with TBST, and incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary Ab (Cell Signaling Technology) for 1 hr at room temperature. Finally, immunoblot signals were visualized using BeyoECL Plus (Beyotime), then imaged and quantitated using a ChemiScope 3400 Mini (CLINX Science Instruments, Shanghai, China).
5
Quantitative Analysis of miRNA and mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TRIzol reagent (Life Technologies) was used for the total RNA extraction. The extracted RNA was then diluted in proportion; and the extracted RNA concentration was measured with a cuvette on the ultrafine nucleic acid quantitative spectrometer (Thermo Nanodrop 1000; Thermo Fisher Scientific). Each sample was repeated three times, and the data was recorded. The reverse transcription was performed as the instructions of the miRNA reverse transcriptase (NCode miRNA First‐Strand cDNA Synthesis Kit; Invitrogen) and reverse transcriptase kit (RevertAid First‐Strand cDNA Synthesis Kit; Thermo Scientific). Real‐time PCR was conducted based on the instructions of the SYBR Green qPCR Master Mix (Fermentas). U6 was set as the endogenous reference of miR‐450b‐5p and MDM2 was that of glyceraldehyde phosphate dehydrogenase (GAPDH). The difference in target gene RNA expression was compared using the 2 ‐ ∆ ∆ C t 20 The RT‐qPCR primers are shown in Supporting Information: Table S1 .
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!