Total cellular RNA was extracted from rat liver using TriZol reagent. RNA concentration was determined spectrophotometrically at 260 nm (Biophotometer plus, Eppendorf, Hamburg, Germany) and the purity of RNA preparation was checked by calculating the absorbance ratio at 260/280 nm. qRT-PCR was conducted in a two-step PCR procedure. Total cellular RNA (2.0 μg) was reverse transcribed by standardized procedure and the transcribed cDNA was quantified (Biophotometer plus, Eppendorf, Hamburg, Germany). qRT-PCR amplification was performed in a 20 μL reaction mixture containing cDNA (100 ng), 1 μL each of 0.3 μM of reverse and forward primers, 10 μL Maxima SYBR green qPCR master mix, and sterile water. The nucleotide sequences of primers used are given in Table 2 . PCR program was conducted using real-time PCR system Mastercycler ep realplex (Eppendorf, Hamburg, Germany) in universal cycling conditions (10 min at 95°C, 40 cycles of 2 min at 95°C, 30 sec at 60°C (or the optimal Tm), and 20 sec at 72°C). The amount of the target gene normalized to an endogenous control glyceraldehyde 3 phosphate dehydrogenase (GAPDH) by 2−ΔΔCT method and the relative quantity was expressed in bar graphs as fold change with respect to control.
Biophotometer plus
Manufactured by Eppendorf
Sourced in Germany, United States, United Kingdom, China, Austria
The BioPhotometer Plus is a compact and versatile UV/Vis spectrophotometer developed by Eppendorf. It is designed to measure the absorbance of samples across a wide range of wavelengths, enabling precise quantification of various biomolecules such as DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (38 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (17 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (10 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (7 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Rna 6000 nano kit, by Agilent Technologies (6 mentions)
Sv total rna isolation system, by Promega (6 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (5 mentions)
Nucleospin rna xs kit, by Macherey-Nagel (5 mentions)
Steponeplus, by Thermo Fisher Scientific (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (5 mentions)
Reliaprep ffpe gdna miniprep system, by Promega (5 mentions)
Sybr green qpcr supermix, by Thermo Fisher Scientific (5 mentions)
Rneasy kit, by Qiagen (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Fenozol, by A&A Biotechnology (4 mentions)
Taqman assay reagent, by Thermo Fisher Scientific (4 mentions)
385 protocols using biophotometer plus
1
Quantitative RT-PCR for Rat Liver Transcripts
2
Spectrophotometric Purity Analysis of Metagenomic DNA
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In the spectrophotometric method, the Eppendorf BioPhotometer Plus instrument was used to determine the purity of the extracted metagenomic DNA from the samples. Measuring procedure was according to literature insert in the manufacturer's manual, with the instrument set for dsDNA and sample dilutions of 5µL sample + 95µL diluent for reading at A260/A280. The Eppendorf BioPhotometer Plus instrument switched-on to initialize. Sample preparation (95µL of diluent distilled water) was pipetted into appropriately labelled tubes. 5µL of mDNA extract was added to the corresponding labelled tubes. The sample was transferred into a clean cuvette shaft of outside diameter 12.5mm x 12.5mm. The instrument was set at blank (zero) before reading at A260/A280 wavelength (Table 4). Result was recorded for purity value at A260/A280 and dsDNA concentration in µg/mL but readings of the dsDNA concentration were disregarded because of inconsistent values (non-reproducibility of readings).
3
Extraction and Quantification of Total Cellular Proteins
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For total cellular protein extraction, the harvested cells were washed twice with cold PBS buffer, and lysed in RIPA buffer (Radio Immunoprecipitation Assay buffer, Beyotime, P0013B) containing protease inhibitor cocktail (Millipore). The protein concentrations were determined by Bradford dye (BIO-RAD, 5000205) in Eppendorf Bio-photometer Plus (Eppendorf). Afterwards, the protein extracts were separated in NuPAGE Bis-Tris gels (Invitrogen), and transferred to PVDF (polyvinylidene difluoride) membranes (Millipore). The membranes were probed by specific antibodies.
4
Isolation and Characterization of sHSP Genes from Bemisia tabaci
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Total RNA was extracted from Bemisia tabaci adults and pupae using the SV Total RNA Isolation and Purification Kit (Promega, Madison, WI, USA). The integrity, purity and concentration of RNA were examined using 1% agarose gel electrophoresis and spectrophotometry at 260 and 280 nm (Eppendorf BioPhotometer plus, Eppendorf, Germany). RNA samples were stored at −80 °C until needed. cDNA was synthesized using an oligo(dT)18 primer (TaKaRa, Dalian, China), and full-length cDNAs of genes encoding sHSPs were obtained by 5′-and 3′-RACE (SMART RACE, Clontech, Mountain View, CA, USA) using the primers shown in Table 1 . Full-length sequences were confirmed by RACE 5′ cDNA.
5
RNA Extraction and cDNA Synthesis
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The RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech, Beijing, China) was used for total RNA extraction according to the manufacturer’s specifications. The concentrations of RNA were quantified using an Eppendorf BioPhotometer Plus (Eppendorf, Hamburg, Germany). Reverse-transcription reactions were performed using a Transciptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol on a T100TM Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, United States).
6
Bacterial DNA Extraction and Purification
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Extraction and purification of DNA
Template DNA was extracted from whole organisms by boiling [22 (link)]. Bacteria were harvested from an overnight broth culture (Biokar Diagnostics, BK015HA, France), suspended in 1 ml sterile Milli-Q water (Milli-Q™, Millipore Corporation, USA). A suspension of 200 μl was incubated at –20 °C for 15 min and boiled at 95 °C for 15 min. The suspension was immediately cooled at 4 °C for 10 min and then centrifuged at 14,000 rpm for 10 min to pellet the cell debris.
The DNA template was purified according to the method described by Zimmermann et al. [23 ]. The purity and DNA concentration of the extract were determined by spectrophotometer (Eppendorf BioPhotometer plus, USA).
Template DNA was extracted from whole organisms by boiling [22 (link)]. Bacteria were harvested from an overnight broth culture (Biokar Diagnostics, BK015HA, France), suspended in 1 ml sterile Milli-Q water (Milli-Q™, Millipore Corporation, USA). A suspension of 200 μl was incubated at –20 °C for 15 min and boiled at 95 °C for 15 min. The suspension was immediately cooled at 4 °C for 10 min and then centrifuged at 14,000 rpm for 10 min to pellet the cell debris.
The DNA template was purified according to the method described by Zimmermann et al. [23 ]. The purity and DNA concentration of the extract were determined by spectrophotometer (Eppendorf BioPhotometer plus, USA).
7
Genomic DNA Extraction and Evaluation
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Genomic DNA was extracted from a 200 μL sample of peripheral blood using a Qiagen DNA Mini kit (Qiagen, Hilden, Germany). The quantity and quality of the DNA samples were evaluated using an Eppendorf BioPhotometer plus (Eppendorf AG, Hamburg, Germany) and automated electrophoresis (Agilent 2200 TapeStation system, Santa Clara, California, United States).
8
Extracting and Analyzing Total RNA
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Total RNA was extracted from all TF samples using the RNeasy Plus Mini Kit (Qiagen, HildenGermany). Five TR samples were also analyzed as standard control isolates. The RNA quality and quantity were analyzed using 1% agarose gel electrophoresis and spectrophotometer (Eppendorf BioPhotometer Plus, Eppendorf, Germany), respectively. Then, cDNA was synthesized using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) with oligo dT and random hexamer primers based on the manufacturer’s instruction.
9
Skin Pigmentation Measurement of Indian Tribes
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The samples from India represent six tribe and caste populations collected from West Maharashtra, which have been described in a prior study (Jonnalagadda, Ozarkar, et al. 2016 (link)). Each participant’s age, sex, native place, and clan was recorded along with 5–8 ml whole blood, which was collected in EDTA vials. Genomic DNA was extracted using phenol–chloroform extraction method (Sambrook et al. 1989 ), and was checked for its quality on a 1% agarose gel. The DNA samples were quantified using the Eppendorf BioPhotometer plus. The final number of samples genotyped was 480.
Constitutive skin pigmentation was measured quantitatively using the DSM II Colormeter (Cortex Technology, Denmark) and recorded in the form of Melanin Index (MI) measures with higher MI values representing darker pigmentation. Three measurements were recorded on the inner surface of both upper arms and were averaged to yield a mean MI value for each study participant.
Constitutive skin pigmentation was measured quantitatively using the DSM II Colormeter (Cortex Technology, Denmark) and recorded in the form of Melanin Index (MI) measures with higher MI values representing darker pigmentation. Three measurements were recorded on the inner surface of both upper arms and were averaged to yield a mean MI value for each study participant.
10
Co-Culture of Yeast Species
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Kluyveromyces marxianus NRRL Y-1109 and Debaryomyces hansenii NRRL Y-1448 were acquired from the culture collection of the United States Department of Agriculture (USDA). The yeasts were seeded separately in 50 mL of LYP in 250 mL flasks. They were incubated at 25 °C and 200 rpm for 20 h in a rotary incubator (INNOVA 44 New Brunswick Scientific). For the fermentation assays, both K. marxianus and D. hansenii were inoculated for an initial OD600 (UV-VIS spectrophotometer, Eppendorf BioPhotometer plus) of 0.5 and 0.1, corresponding to initial cell densities of 5.0 × 107 and 1.0 × 107 CFU/mL, respectively. This co-culture ratio was selected based on a previous study (Valdez Castillo et al., 2021) [11 (link)].
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