The mRNA levels of TCF4 and β-catenin were quantified by RTPCR. Briefly, total RNA was extracted from the tissue samples using RNAiso Plus (Takara, Cat. #9108). The integrity of total RNA was checked electrophoretically and RNA concentration was quantified with a NanoDrop spectrophotometer. Two microgram of total RNA was used for the synthesis of complementary DNA (cDNA) using the cDNA Synthesis Kit (Thermo Scientific, #K1622) and amplified using the TopTaq DNA Polymerase (Qiagen, Cat. # 200201) according to the manufacturer's instruction. All primers were analyzed using Primer-Blast to ensure primer specificity for the gene of interest (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences for TCF4 forward 5'-GAATCGTCCCAGAGTGATGTCG-3' and reverse 5'-TGCACTCAGCTACGACCTTTGC-3'. The primer sequences for β-catenin forward 5'-CACAAGCAGAGTGCTGAAGGTG-3' and reverse 5'-GATTCCTGAGAGTCCAAAGACAG-3'. The primer sequences for reference gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) forward 5'-GTCTCCTCTGACTTCAACAGCG-3' and reverse 5'-ACCACCCTGTTGCTGTAGCCAA-3'. The PCR products were run on 1% w/v agarose gels, stained with ethidium bromide (0.5 μg/ml), and photographed by a BioRad Gel Doc™ EZ (Bio-Rad Laboratories, Hercules, CA, USA). The intensity of bands was noted from Image Lab version 5.2.1 software of BioRad Gel Doc™ EZ.
Biorad gel doc ez
Manufactured by Bio-Rad
Sourced in United States
The BioRad Gel Doc™ EZ is a compact and automated imaging system designed for the analysis of gel-based samples. It provides high-quality digital images of various types of gels, including agarose and polyacrylamide gels, as well as stained blots. The system utilizes LED illumination and a sensitive CCD camera to capture images, which can then be analyzed using the accompanying software.
Automatically generated - may contain errors
Lab products found in correlation
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Pcr pre mixture, by Bioneer (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Toptaq dna polymerase, by Qiagen (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Gelcompar 2 version 6, by bioMérieux (1 mentions)
4 protocols using biorad gel doc ez
1
Quantifying TCF4 and β-Catenin mRNA Levels
2
Semi-quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from control and daurinol treated cells using TRIzol reagent (Ambion, Austin, TX, USA). For semi-quantitative RT-PCR, 1 μg of RNA was used as a template for reverse transcription using the Prime Script 1′st strand cDNA Synthesis kit (Takara; Kyoto, Japan). PCR was carried out with 20 ng of cDNA using a PCR pre-mixture (Bioneer, Daejeon, Korea). The sequences of primers used were as follows: GAPDH (forward: 5′-TTTGGTCGTATTGGGCGCCTG-3′; Reverse: 5′-CCATGACGAACATGGGGGCAT-3′), MMP2 (forward: 5′-TCGCCCATCATCAAGTTC-3′; Reverse: 5′-GTGATCTGGTTCTTGTCC-3′), MMP9 (forward: 5′-AACCAATCTCACCGACAG -3′; Reverse: 5′-CAAAGGCGTCGTCAATCA-3′), and uPA (forward: 5′-CCAATTAGGAAGTGTAAGCAGC-3′; Reverse: 5′-GCCAAGAAAGGGACATCTATG-3′). PCR products were separated on 1.5% agarose gel stained with NEOgreen (Insungscience, Seoul, Korea) and visualized under UV lighting using Bio-Rad Gel Doc EZ (Bio-Rad, Hercules, CA, USA).
3
SDS-PAGE Analysis of Milk Thistle Protein
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The SDS‐PAGE analysis was carried out in accordance with Laemmli's method (Laemmli, 1970 (link)). Milk thistle protein extract was dissolved in distilled water at a concentration of 10 mg/mL. The extract solution was mixed with sample buffer (0.5 M Tris–HCl pH 6.8, glycerol, 10% SDS, w/v, 0.5% bromophenol blue, β mercaptoethanol) at a ratio of 1/1 (v/v). It was then heated at 95°C for 3 min. The electrophoresis tank was filled with electrolyte buffer, including 25 Mm Tris, 0.1% SDS, and 192 mM glycine. 10 μL of sample was loaded onto 4% stacking gel (w/v) and 7.5–30% gradient polyacrylamide gel (w/v) with the help of a microsyringe. The gel was electrophoresed vertically using a Mini‐PROTEAN®System (Bio‐Rad Laboratories, Inc, USA) at a constant current of 20 mA. The gel was subsequently dyed with a solution containing 0.25% (w/v) Coomassie Brilliant Blue is a type of dye. A mixture of water/methanol/acetic acid (6/3/1, v/v/v) was used in the dye removal process. The gel was photographed with an imaging system (Biorad Gel Doc EZ, Bio‐Rad Laboratories Inc, USA).
4
Bacterial Strain Fingerprinting via GTG5-PCR
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Fingerprinting PCR was carried out in 20µL reaction tubes in a Bio-Rad T100™ Thermal Cycler (Bio-Rad, Singapore, Singapore). The reaction mixture consisted of 10µL KAPA BIOSYSTEMS 2X KAPA Taq Ready Mix (Kapa Biosystems Cape Town, South Africa), 0.3µL GTGGTGGTGGTGGTG oligonucleotide primer (Versalovic et al., 1994) , 2µL DNA template, 7.3µL PCR-grade water and 0.4µL Dimethyl sulfoxide (DMSO). The PCR program was as follows: 95 o C for 5 min, 95 o C for 1 min, annealing at 52 o C for 1 min, extension at 72 o C for 3 min. The program was repeated for 34 cycles and a final extension at 72 o C for 10 minutes. PCR products were separated by Gel electrophoresis using a 1.5 % Agarose gel (55V for 4h) and the image viewed using a Bio-Rad Gel Doc™ EZ (Bio-Rad, California, USA). The GTG5 fingerprints were analysed using Gel-Compar II version 6.5 (Applied Maths NV, Sint-Martens-Latem, Belgium). The similarity of digitised bands patterns was calculated using Pearson's correlation coefficient, and unweighted pair group method with arithmetic means. Complete linkage algorithms were used to construct an average linkage dendrogram to show relationship of isolates. Isolates were considered to be within a clonal cluster if relatedness was 70% and above (Stackebrandt et al., 2002) . However, due to the close similarities that existed between the isolates sub clusters were further considered at 95%.
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