RNA was extraction was performed using the Isolate II RNA extraction kit (Bioline, cat # BIO-52073) according to the manufacturer’s protocol. RNA concentration was determined using a NanoPhotometer® NP80 spectrophotometer (Implen). cDNA was synthezised from approximately 200 ng of RNA using the iScript gDNA clear cDNA synthesis kit (Bio-Rad, cat # 172-5035) according to the manufacturers’ instructions. qRT-PCR was performed with the QuantiTect SYBR Green PCR Kit (Qiagen, cat # 204143) using a CFX384 Real Time PCR System (Bio-Rad) with DKK1 (Qiagen, Geneglobe ID # QT00009093), LEF1 (Qiagen, Geneglobe ID # QT00021133) or RRN18S (Qiagen, Geneglobe ID # QT00199367) primers, and analyzed using CFX Manager version 3.1 software (Bio-Rad). The relative expression of DKK1 or LEF1 were normalized to the loading control gene RRN18S, and quantified using the ΔΔCT method.
Nanophotometer np80 spectrophotometer
Manufactured by Implen
Sourced in Germany
The NanoPhotometer® NP80 is a compact, single-beam UV-Vis spectrophotometer designed for accurate measurement of nucleic acid and protein concentrations. It features a wavelength range of 200-800 nm and supports small sample volumes down to 0.5 μl.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Iscript gdna clear cdna synthesis kit, by Bio-Rad (1 mentions)
Isolate 2 rna extraction kit, by Meridian Bioscience (1 mentions)
Cfx manager version 3, by Bio-Rad (1 mentions)
Cfx384 real time pcr system, by Bio-Rad (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Plant genomic dna extraction kit, by Tiangen Biotech (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
6 protocols using nanophotometer np80 spectrophotometer
1
Gene Expression Quantification via qRT-PCR
2
Leishmania spp. DNA Extraction Protocol
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The biological samples were initially seeded at 26 °C in Schneider’s Drosophila medium supplemented with 20% heat-inactivated fetal bovine serum (FBS) and an antibiotic cocktail (50 U/mL penicillin, 50 mg/mL streptomycin) in order to facilitate the isolation and growth of the parasites.
Three different Leishmania spp., L. donovani LV9, L. infantum BCN 150 (MCAN/ES/96/BCN150) and L. guyanensis, were used as reference and controls. L. donovani promastigotes were kindly provided by Philippe M. Loiseau from the Université Paris-Sud, BCN 150 by Prof. Dr. Rafael Balaña Fouce, from the Instituto de Biotecnología de León (INBIOTEC) and L. guyanensis were purchased from the American Type Culture Collection (ATCC).
The parasites were collected by centrifugation and the DNA was extracted by following the protocol described by Medina-Acosta et al. [20 (link)]. The samples were washed with ethanol and finally purified with RNase A (0.1 mg/mL) for 1 h at 37 °C and proteinase K (0.14 mg/mL) for 1 h at 60 °C. DNA concentrations were measured on a NanoPhotometer NP80 spectrophotometer (Implen, Munich, Germany).
Three different Leishmania spp., L. donovani LV9, L. infantum BCN 150 (MCAN/ES/96/BCN150) and L. guyanensis, were used as reference and controls. L. donovani promastigotes were kindly provided by Philippe M. Loiseau from the Université Paris-Sud, BCN 150 by Prof. Dr. Rafael Balaña Fouce, from the Instituto de Biotecnología de León (INBIOTEC) and L. guyanensis were purchased from the American Type Culture Collection (ATCC).
The parasites were collected by centrifugation and the DNA was extracted by following the protocol described by Medina-Acosta et al. [20 (link)]. The samples were washed with ethanol and finally purified with RNase A (0.1 mg/mL) for 1 h at 37 °C and proteinase K (0.14 mg/mL) for 1 h at 60 °C. DNA concentrations were measured on a NanoPhotometer NP80 spectrophotometer (Implen, Munich, Germany).
3
RNA Extraction from C. elegans Samples
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RNA was purified from snap-frozen packed C. elegans pellets using TRIzol Reagent as directed by the manufacturer’s instructions51 (link). RNA was resuspended in 50 uL sterile water. RNA concentration and purity were assessed using a NanoPhotometer® NP80 spectrophotometer (Implen GmbH, Munich, Germany). RNA integrity was assessed by 1% Tris/Borate/EDTA (TBE) agarose gel electrophoresis.
4
RAD-seq Library Preparation from F2:3 Plants
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Young leaves from F 2:3 individuals and their parents were collected and used to extract genomic DNA following the manufacturer's protocol of the Plant Genomic DNA extraction Kit (Tiangen Biotech, Beijing, China). DNA concentration and purity were assessed using a NanoPhotometer® NP 80 spectrophotometer (IMPLEN, CA, USA).
The RAD-seq library was developed based on a previously described protocol (Baird et al. 2008; Guo et al. 2015) . Genomic DNA of the F 2:3 strains and their parents were digested by EcoRI (New England Biolabs, Ipswich, MA, USA) and were then ligated to a P1 adapter containing a nucleotide barcode for individual labeling. The digested DNA and the P1 adapter from different samples were pooled together and randomly sheared. The 400-700 bp fragments were selected and ligated with the P2 adapter. Several rounds of PCR ampli cation were performed to enrich the adapter-ligated DNA fragments, and DNA fragments within the 400-700 bp range were selected and puri ed for library construction. The quali ed library was sequenced on HiSeq4000 platform using the PE150 strategy.
The RAD-seq library was developed based on a previously described protocol (Baird et al. 2008; Guo et al. 2015) . Genomic DNA of the F 2:3 strains and their parents were digested by EcoRI (New England Biolabs, Ipswich, MA, USA) and were then ligated to a P1 adapter containing a nucleotide barcode for individual labeling. The digested DNA and the P1 adapter from different samples were pooled together and randomly sheared. The 400-700 bp fragments were selected and ligated with the P2 adapter. Several rounds of PCR ampli cation were performed to enrich the adapter-ligated DNA fragments, and DNA fragments within the 400-700 bp range were selected and puri ed for library construction. The quali ed library was sequenced on HiSeq4000 platform using the PE150 strategy.
5
Total RNA Extraction and Purification
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Total RNA was isolated from the kidneys, blood, and liver with a TRI reagent (Sigma Aldrich, Taufkirchen, Germany). RNA was purified from DNA with a DNA-free kit (Ambion, Austin, TX, USA) and its quantity and purity determined with a NanoPhotometer ® NP 80 spectrophotometer (ImplenGMBH, Munich, Germany).
The quality of RNA was analysed with gel electrophoresis. cDNA was synthesised from the obtained RNA (5 μg from kidneys and liver, and 2 μg from blood) with random hexamer primers (RevertAid™ First Strand cDNA Synthesis Kit, Fermentas, Vilnius, Lithuania).
The quality of RNA was analysed with gel electrophoresis. cDNA was synthesised from the obtained RNA (5 μg from kidneys and liver, and 2 μg from blood) with random hexamer primers (RevertAid™ First Strand cDNA Synthesis Kit, Fermentas, Vilnius, Lithuania).
6
RNA Extraction from C. elegans Samples
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RNA was extracted from L2 and L4 snap-frozen C. elegans pellets using TRIzol Reagent (Thermo Fisher Scientific, Inc.) per manufacturer’s protocol. RNA was resuspended in 50 μL sterile water and concentration was assessed using NanoPhotometer® NP80 spectrophotometer (Implen GmbH).
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