Amplicons from steps 1 and 2 PCR assays were run in 2% agarose gels (Promega, USA) with 15% GelRedTM nucleic acid gel stain (Biotium, USA) at 280 V for 30 min (Bio-Rad, USA) using 1× Tris-Acetic Acid-EDTA (TAE) as running buffer. Amplicons from steps 3 and 4 PCR assays were run in 3% agarose gels (Promega, USA) with 15% GelRedTM nucleic acid gel stain (Biotium, USA) at 280 V for 50 min (Bio-Rad, USA). Amplicon sizes were estimated using 100-bp plus molecular weight marker (Kapa Biosystems, USA). Gels were viewed under UV using a gel documentation system (Vilber Lourmat, France).
Gelred nucleic acid gel stain
Manufactured by Biotium
Sourced in United States
GelRed Nucleic Acid Gel Stain is a fluorescent dye designed for staining DNA and RNA in agarose and polyacrylamide gels. It is a highly sensitive and stable stain that can detect as little as 0.1 ng of nucleic acid per band.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
T100 thermal cycler, by Bio-Rad (5 mentions)
Gotaq green master mix, by Promega (5 mentions)
Platinum pcr supermix, by Thermo Fisher Scientific (4 mentions)
Gel doc xr system, by Bio-Rad (4 mentions)
Generuler 100 bp plus dna ladder, by Thermo Fisher Scientific (4 mentions)
Qiaquick gel extraction kit, by Qiagen (4 mentions)
Gel doc xr gel documentation system, by Bio-Rad (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Onestep rt pcr kit, by Qiagen (3 mentions)
Tmoimr1594, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Molecular imager gel doc xr, by Bio-Rad (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Proteinase k, by Roche (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
213 protocols using gelred nucleic acid gel stain
1
Agarose Gel Electrophoresis of PCR Amplicons
2
Genotyping of UCP2 Polymorphisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from peripheral blood leucocytes by a standardized salting-out procedure. The UCP2 -866G/A polymorphism (rs659366) was determined by digesting polymerase chain reaction (PCR) products with the restriction enzyme MluI (Invitrogen Life Technologies, Inc., CA, USA), as previously described [23 (link)]. Digestion fragments were resolved on 2% agarose gels containing GelRed Nucleic Acid Gel Stain (Biotium, Inc., Hayward, CA) and visualized under ultraviolet illumination. A sample of DNA (whose genotype was identified by sequencing) was used as a positive control to evaluate the completeness of PCR product digestion. Evaluation of the UCP2 45 bp Ins/Del polymorphism was done by PCR, as previously described [23 (link)]. Briefly, primers amplified products of 457 bp (insertion allele) or 412 bp (deletion allele), which were resolved on 2.5% agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium, Inc.) and visualized under ultraviolet light. Genotypes of the -866G/A and Ins/Del polymorphisms were recorded using the ImageMaster System VDS (GE HealthCare, London, UK).
Genotyping of the UCP2 Ala55Val (C/T) polymorphism (rs660339) was determined using primers and probes contained in the Human Custom TaqMan Genotyping Assay 40x (Life Technologies, Foster City, CA, USA). Primer and probe sequences can be found elsewhere [23 (link)].
Genotyping of the UCP2 Ala55Val (C/T) polymorphism (rs660339) was determined using primers and probes contained in the Human Custom TaqMan Genotyping Assay 40x (Life Technologies, Foster City, CA, USA). Primer and probe sequences can be found elsewhere [23 (link)].
3
Cell Viability Assessment of Withaferin A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was determined by flow cytometry through addition of 1× GelRed™ Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). MDA-MB-231 and MCF-7 cells were plated in 24-well plates and treated with 6 different concentrations of WA (0.0875, 0.175, 0.35, 0.7, 1.4, 2.8 µM) or mock control (0.014% DMSO) as described above. After the indicated time point, cell growth medium, PBS wash solution as well as harvested cells were collected and centrifuged for 5 min at 250 x g. The cell pellet was resuspended in 1 mL of PBS containing 5% FBS. For each concentration, three replicates were performed and directly analyzed using an Epics XL-MCL analytical flow cytometer (Beckman Coulter, Fullerton, CA, USA). The mean value of at least three independent experiments was calculated. IC50 concentrations were estimated for 72 h treatments using GraphPad Prism version 5.00 for Windows, modeling a dose response curve starting from 100% viability in DMSO controls to 0% viability at the highest concentrations of WA. The hill slope was not fixed.
4
Amplification and Visualization of Hymenopteran HVR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The csd HVR was amplified using a primer pair reported by Hyink et al. [28 (link)], also used by Zareba et al. [23 (link)]: forward: 5′-TATCGAGAAAsATCGAAAGAACGAT-3′, reverse: 5′-ATTGAAATCCAAGGTCCCATTGGT-3′. Amplifications were performed on a 2700 Thermal Cycler (Life Technologies, Waltham, MA, USA). Reactions were run in a total volume of 20 μL including KAPA HiFi HotStart Mastermix (Roche, Basel, Switzerland); 10 pmol of each primer; 40 ng of template DNA. The PCR profile was the following: initial denaturation step at 95 °C for 3 min; 35 cycles of alternate temperatures (20 s at 98 °C, 15 s at 51 °C, 30 s at 72 °C); and a final extension step at 72 °C for 1 min. Obtained amplicons were electrophoresed on 2.5% agarose gels in TBE 1× buffer and then visualized with 1× GelRed Nucleic Acid Gel Stain (Biotium Inc., Hayward, CA, USA).
5
Multiplex PCR for Salmonella Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The multiplex PCR assays were performed in a final volume of 25-μL including 200 μM deoxynucleoside triphosphate mix, 0.25 μL (1 U) of Taq DNA polymerase, 2.5 μL of 10× Taq DNA polymerase buffer (Vazyme, Nanjing, China), 40 nM of the ratA ROD F/R primers, 80 nM of the I137_08605 F/R primers, 80 nM of the stn F/R primers, and 100 ng of the bacterial genomic DNA. The stn gene encodes Salmonella enterotoxin and is specific for S. enterica (Prager et al., 1995 (link)). Therefore, stn could be used as the internal amplification control in the multiplex PCR method. The PCR amplification was conducted using a T100 Thermal Cycler (Eppendorf, Hamburg, Germany) with the following protocol: initial denaturation at 94°C for 5 min; 30 sequential cycles of 94°C for 45 s, 52°C for 45 s, and 72°C for 40 s; and a final step of 72°C for 10 min. The PCR fragments were separated using 1% agarose gels after electrophoresis, and stained with 1× GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, United States). The amplified PCR products were visualized using UV light excitation, and images were digitalized using a GelDoc XR Gel Documentation System (Bio-Rad).
6
Genotyping Mouse Pups Using PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse pups were genotyped using a PCR test with tail DNA. Briefly, PCR of tail DNA was performed according to the protocol offered by the Jackson Laboratory utilizing oligonucleotide primers oIMR3610 (AGG ACT GAC CAC TCG ACC AG) and oIMR3611 (CGG GGG TCT AGT TCT GCA T) for APP and oIMR1644 (AAT AGA GAA CGG CAG GAG CA) and oIMR1645 (GCC ATG AGG GCA CTA ATC AT) for PSEN1, purchased from Invitrogen (Carlsbad, CA, USA). The PCR reaction mixture included 1 µL DNA template and 23 µL Platinum PCR SuperMix (Invitrogen) complemented with 0.39 µM of each primer (Invitrogen); the entire volume was 25 µL. The following cycling conditions were employed: 5 min at 95 °C, 35 cycles at 30 s at 95 °C, 30 s at 56 °C, 60 s at 72 °C, and 10 min at 72 °C. Reaction products were evaluated on a 1.2% agarose gel subjected to 100 V for 60 min using Tris–acetate–EDTA running buffer, including 1× GelRedTM Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA).
7
Intron Amplification by Gradient PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To amplify each intron region, gradient PCR was used to optimize suitable conditions using an annealing temperature ranging between 50 and 60 °C. The PCR conditions were initial denaturation at 94 °C for 30 s, annealing at 50–60 °C for 30 s, extension at 72 °C for 1 min, and a final extension 72 °C for 5 min. In the case of low concentration (no/faint band), one microliter of the first PCR product was used as the DNA template for the second PCR using the same conditions as used with the first PCR. The PCR mixture contained 1× TaKaRa Ex PCR buffer, 0.2 mM dNTPs (each), 0.2 μM of each primer, and 1.0 U of TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). Subsequently, the PCR product underwent electrophoresis in 1% agarose gel and was visualized with GelRedTM Nucleic Acid Gel Stain (Biotium, Inc., Hayward, CA, USA). The PCR product (~1000 bp) was cut for gel purification using an E.Z.N.A.® Gel Extraction kit (Omega bio-tek, Norcross, GA, USA) and was subsequently used for DNA sequencing and cloning.
8
Quantitative RT-PCR Analysis of Sqstm1 and Gapdh
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted with the RNeasy kit (Qiagen, 74004). One microgram of total RNA from each sample was used as a template for cDNA synthesis with a QuantiTect reverse transcriptase kit (Qiagen, 205311). An equal volume of cDNA product was used in the PCR performed using the TopTaq Master Mix Kit (Qiagen, 200403). PCR amplification was performed using the following primers (purchased from Invitrogen): mouse Sqstm1 gene 5′-AGCTGCCCTC AGCCCTCTA’-3 (forward) and 5′-GGCTTCTCTT CCCTCCATGTT’-3 (reverse) and Gapdh 5′-ATGGTGAAGG TCGGTGTGAA’-3 (forward) and 5′ TTCACACCCA TCACAAACAT'-3 (reverse). The PCR conditions were set according to the standard protocol. The PCR products were resolved on an agarose gel containing GelRedTM nucleic acid gel stain (Biotium, 41002) and exposed using a Kodak Image Station 440CF (Eastman Kodak Company, New Haven, CT USA).
9
Quantification of Gene Expression in Bovine Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA isolation from liver biopsies, cDNA synthesis and quantitative real-time polymerase chain reaction (qPCR) were carried out as described recently in detail [34 (link)]. Expression values of the genes investigated were normalised using the GeNorm normalisation factor [57 ]. Procedure of normalisation and average expression stability ranking of the six potential reference genes in liver of cows were also performed as described recently [34 (link)]. The characteristics of gene-specific primers are shown in Table 4 . After normalisation of gene expression data using the calculated GeNorm normalisation factor, means and SEM were calculated from normalised expression data for samples of the same treatment group. The mean of 3 wk antepartum was set to 1 and relative expression ratios of 1, 5 and 14 wk postpartum are expressed as fold changes compared to 3 wk antepartum. To determine the expression of spliced and unspliced XBP1, the PCR run was stopped within the linear range of amplification. Subsequently, the PCR products were separated using 2% agarose gel electrophoresis stained with GelRedTM nucleic acid gel stain (Biotium, California, USA), visualized under UV light, and digitalized with a digital camera (SynGene, Cambridge, England).
10
Genetic Diversity of Chorthippus cazurroi
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We extracted DNA from the hind femur or head using the Qiagen DNeasy kit (Venlo, Netherlands) and following the manufacturer’s protocol. To determine genetic variability of Chorthippus cazurroi, we amplified a 575 bp fragment of the cytochrome oxidase subunit I (COI) using the primers LCO1490 and HCO2198 [38 ]. Polymerase chain reactions (PCR) were set up in 10 µl total volumes following the procedures and conditions described in previous studies [36 (link),39 (link)]. PCR products were checked by electrophoresis on 1.5% agarose gels stained with GelRedTM nucleic acid gel stain (Biotium, Inc., Hayward, CA, USA). We performed sequencing reactions using the Perkin Elmer BigDye v. 3.1 (Applied Biosystems, Carlsbad, CA, USA) terminator reaction mix in a volume of 10 µL, following the procedures and conditions described in Pato et al. [39 (link)]. The final product was purified and sequenced on an ABI PRISM® 3130xl Genetic Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequences were edited and aligned by hand using BioEdit v. 7.2.5 [40 ]. Unique sequences were deposited in the GenBank database (NCBI) with the accession numbers MH271324–MH271356.
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!