Total RNA was extracted from the EPDENs and plant tissues using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was resuspended in 100 µl of DEPC-treated water. The purified RNA was twice digested with DNase I (Qiagen). The purity and yield of RNA was determined spectrophotometrically by measuring optical density at wavelengths of 260 and 280 nm. Samples were stored at −80°C. To confirm that the nucleic acid isolated from EPDENs was RNA, nucleic acid from EPDENs was treated with 1.0 µg/µl RNase (Sigma) or DEPC treated water as a control for 15 min at 37°C before the samples were loaded on a 12% polyacrylamide gel. A total of 1 µg RNA isolated from EPDENs was resolved on 12% polyacrylamide (acrylamide/bis-acrylamide, 29:1) gels containing 8 M Urea and 1×Tris-Boric Acid-EDTA (TBE, 89 mM tris (pH 7.6), 89 mM boric acid, 2 mM EDTA). A total of 1 µg RNA isolated from plant tissue extracts were resolved on 2% agarose gel. After electrophoresis, the gel was stained with ethidium bromide (0.5 µg/ml) and visualized using a UVP PhotoDoc-It™ Imaging System (UVP, Montpelier, Maryland).
Photo doc it imaging system
Manufactured by Analytik Jena
Sourced in United States
The Photo Doc-IT Imaging system is a laboratory equipment designed for capturing high-quality images of gels, blots, and other samples. It provides a reliable and efficient imaging solution for various applications in life science research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Tbe buffer, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Seakem le agarose, by Cambrex (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Ds 11, by DeNovix (1 mentions)
100 bp dna ladder marker, by Promega (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Gotaq flexi dna polymerase, by Promega (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
7 protocols using photo doc it imaging system
1
Total RNA Extraction and Analysis
2
Restriction Enzyme Digestion of 5'UTR
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Two combinations of restriction endonucleases; MvaI/HinfI and RsaI/HaeIII, were used to digest 5′UTR PCR products. Digested products were run side by side with undigested PCR products in 2.5% agarose gels for 40 min in TBE buffer (Thermo Scientific, USA) buffer, stained with Ethidium Bromide and visualized in Photo Doc-IT Imaging system (UVP, USA).
3
DNA Extraction from Plant Flour
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DNeasy plant mini kit (Qiagen, Hilden, Germany) was chosen as a suitable procedure for DNA extraction based on the results of our previous study [33 (link)]. Genomic DNAs were isolated and purified from 100 mg flour. The purity and concentration of the extracted DNAs were estimated by spectrophotometer DeNovix DS-11 (DeNovix Inc. Wilmington, NC, USA). The quantity and integrity of DNA were evaluated using electrophoresis (VWR International, Radnor, PA, USA) on 1% agarose gel (SeaKem LE agarose; Cambrex, East Rutherford, NJ, USA) containing 1 μg/mL of ethidium bromide (Sigma-Aldrich, St. Louis, MI, USA). The agarose gels were visualized under ultraviolet (UV) light and a digital image was obtained using a gel documentation system PhotoDoc- It imaging system (UVP, Upland, CA, USA).
4
Antimicrobial Susceptibility of E. coli Strains
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Wild-type and Δdam-mutant strains of E. coli CFT073 and cured C119 as well as control strains E. coli strain Lo QnrA+ and E. coli JM109 harboring pGEMQA were subjected to antimicrobial susceptibility testing using Sensititre Substrate-in-Well GNUR2F Gram-negative MIC plates (TREK Diagnostic Systems, Inc., OH, USA) for inoculation and incubation (63 (link)). A 50 μl suspension of the sample was used to inoculate Sensititre plates, sealed and incubated at 35°C for 18–24 h. The plates were observed for the presence of a growth button at the base of the microtiter well and fluorescence intensity (+++ to 0) captured with a UV Benchtop Variable Transilluminator and Photo Doc-It Imaging System (UVP, CA, USA).
5
Restriction Enzyme Profiling of 5' UTR
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PCR products of the 5′ UTR region were digested using two combinations of restriction endonucleases; MvaI/HinfI and RsaI/HaeIII. Restriction reactions were run in 2.5% agarose gels for 40 min in TBE (#B52, Thermo Scientific, Waltham MA, USA) buffer, stained for 25 min with Ethidium Bromide and the gels were documented in Photo Doc-IT Imaging system (UVP, Upland CA, USA).
6
Mitochondrial DNA-Based PCR Assay for Meat Species Identification
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Four oligonucleotide primer sets (synthesized by Macrogen Inc. Korea) derived from the mitochondrial DNA cyt b gene sequences for the amplification of chicken, beef, and pork, as designed by Matsunaga et al. [15] , were used for the PCR amplification. The primer sequences are as given in Table 2. Simplex PCR amplification was accomplished in a 25 µl total volume containing 0.625U GoTaq Flexi DNA Polymerase (Promega, Madison, USA), 5 µl of 5X GoTaq Flexi Buffer, 200 µM each of dNTP, 1.5 mM MgCl2, 0.4 µM primers and 1 µl (30 ng/µl) of total DNA in thermal cycler GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA) and the cycling parameters were initial denaturation at 94 o C for 3 min following 35 cycles of denaturation at 94 o C for 30s, annealing at 60 o C for 30s and elongation at 72 o C for 30s and final elongation at 72 o C for 3 min.
Five µl of the PCR products from each amplified product was electrophoresed along with a 100-bp DNA ladder marker (Promega) to confirm the targeted PCR amplification. The electrophoresis was performed on 2% agarose gel containing ethidium bromide (0.5 µg/ml) at constant 80 V for 60 min in 1X Tris-acetate EDTA (TAE) buffer. The bands in the gel were then visualized using PhotoDoc-It Imaging System (UVP)
Five µl of the PCR products from each amplified product was electrophoresed along with a 100-bp DNA ladder marker (Promega) to confirm the targeted PCR amplification. The electrophoresis was performed on 2% agarose gel containing ethidium bromide (0.5 µg/ml) at constant 80 V for 60 min in 1X Tris-acetate EDTA (TAE) buffer. The bands in the gel were then visualized using PhotoDoc-It Imaging System (UVP)
7
Integrin Expression in pTr2 Cells
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Total cellular RNA was isolated from pTr2 cells using an RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Complementary DNA was prepared using 3 µg total RNA with the SuperScript III First-Strand Synthesis System (Life Technologies) according to the manufacturer's instructions. After cDNA synthesis, PCR was performed using 50 ng/reaction of cDNA with a recombinant Taq DNA polymerase (Life Technologies) according to the manufacturer's instructions. Primers for integrin subunits ITGA5, ITGAV, ITGA2, ITGA4, ITGB1, ITGB3, ITGB6 and GAPDH (positive control; Supplementary Table 2, see section on supplementary data given at the end of this article) were derived from conserved porcine sequences and designed using Primer3 (primer3.sourceforge.net/) and produced by Eurofins MWF Operon (Huntsville, AL). The PCR products were analyzed on 1% (wt/vol) agarose gels, and the gels were imaged using the PhotoDoc-It imaging system (UVP, Upland, CA).
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