Twenty-thousand B16 cells were plated in a 12-well plate (0.88 mL of cell suspension) and transfected with 10 nM siRNA (diluted in 120 μL of Opti-MEM containing 3 μL of Lipofectamine RNAiMAX). After 24 h, the 1 mL of transfection mix was replaced with 0.88 mL of normal growth media and, after a further 24 h, cells were again transfected with 10 nM siRNA. After 48 h, the transfection mix was removed and cells were fixed with 2% PFA for 10 min. Cells were stained with 0.1% crystal violet for 10 min, washed, air-dried, and scanned using a Fuji FLA-5100 imager.
Fla 5100 imager
Manufactured by Fujifilm
The FLA-5100 imager is a versatile lab equipment product from Fujifilm. It is designed for high-resolution imaging of various samples, including gels, blots, and autoradiographs. The FLA-5100 utilizes a laser-based detection system to capture and digitize images with high sensitivity and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey sa infrared imaging system, by LI COR (1 mentions)
Multi gauge software version 3, by Fujifilm (1 mentions)
Nupage lds buffer, by Thermo Fisher Scientific (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Sc56766, by Santa Cruz Biotechnology (1 mentions)
Maxiscript t7 kit, by Thermo Fisher Scientific (1 mentions)
N6 methyl atp, by TriLink (1 mentions)
Ribonuclease protection assay 3 kit, by Thermo Fisher Scientific (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Hape1, by New England Biolabs (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
Nuclease p1, by Merck Group (1 mentions)
Pei cellulose tlc plate, by Merck Group (1 mentions)
T4 pnk, by Thermo Fisher Scientific (1 mentions)
7 protocols using fla 5100 imager
1
B16 Cell Transfection Optimization
2
Evaluating Melanoma Cell Viability with A939572 and Oleic Acid
3
AtPol λ Activity Assay
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Reactions (10 μl) contained 50 mM Tris HCl pH 7.5, 1 mM DTT, 0.2 mg/ml BSA, 2% glycerol, 10 mM MgCl2, 100 nM DNA substrate, the indicated amount of AtPol λ and dCTP, dNTPs (deoxynucleotides) or ddNTPs (dideoxynucleotides). When indicated, AtPol λ was pre-incubated with pre-immune serum or anti-AtPol λ antibody for one hour at 4 °C. After incubation at 37 °C for the indicated times, reactions were stopped by adding 20 mM EDTA, 0.6% SDS and 0.5 mg/ml proteinase K, and mixtures were incubated at 37 °C for 30 min. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated at -20 °C in the presence of 0.3 mM NaCl and 16 mg/ml glycogen. Samples were resuspended in 10 µl of 90% formamide and heated at 95 °C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labelled DNA was visualized using FLA-5100 imager (Fujifilm) and analyzed using Multi Gauge software version 3.0 (Fujifilm).
4
Western Blot Analysis of σE and RpoB
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Strains of interest were harvested at mid-exponential phase (OD 0.5), lysed directly in 1X NuPAGE LDS buffer (Novex) containing 6uM DTT, separated by NuPAGE Bis-Tris gel electrophoresis and transferred onto nitrocellulose using the iBlot system (Life Technologies). Blots were incubated with rabbit polyclonal antisera against σE or monoclonal antibody against RpoB (sc56766, Santa Cruz Biotechnology) in 5% milk in TBST. Horseradish-peroxidase conjugated secondary antibodies (Pierce) and Supersignal West Pico chemilumeniscent substrate (Pierce) were used to detect primary antibody signal. Blots were visualized on X-ray film, which was subsequently digitized on a FujiFilm FLA-5100 imager, and bands quantitated using MultiGuage V3.1 image analysis software.
5
Analyzing Actin mRNA Localization by RPA
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This assay was conducted using Ribonuclease Protection Assay (RPA) III kit (Ambion). A 250 nt of mouse β-actin antisense RNA (actin-as-250) was radiolabeled by transcribing pTRI-Actin-Mouse vector provided by RPA kit using T7 Maxiscript Kit (Ambion) with the presence of α-32P-UTP. A 140-bp sense fragment complementary to the probe was amplified by PCR reaction using pTRI-Actin-Mouse vector as template and forward primer 5′-CGGggtaccgacggccaggtcat
cactat-3′ and reverse primer 5′-ATAAGAATGCGGCCGCcggatgtca
acgtcacactt-3′ containing a T7 promoter; 140 nt mouse β-actin sense RNA (actin-s-140) was in vitro-transcribed using T7 Maxiscript Kit (Ambion) with the presence of either ATP or N6-methyl-ATP (Trilink) and was purified by polyacrylamide gel electrophoresis (PAGE). Actin-as-250 was mixed with actin-s-140-m6A or actin-s-140-A at a molecular ratio 5:1 and coprecipitated. RNA pellets were resuspended in hybridization buffer, aliquoted, and incubated for 0.5, 2 and 4 h for sense–antisense annealing and duplex formation. After incubation, unhybridized RNA were digested by RNases A and T1. RNA samples were precipitated and then separated on a 5% acrylamide denaturing gel (National diagnostics). The gel was exposed to a storage phosphor screen and scanned by FujiFilm FLA-5100 imager.
cactat-3′ and reverse primer 5′-ATAAGAATGCGGCCGCcggatgtca
acgtcacactt-3′ containing a T7 promoter; 140 nt mouse β-actin sense RNA (actin-s-140) was in vitro-transcribed using T7 Maxiscript Kit (Ambion) with the presence of either ATP or N6-methyl-ATP (Trilink) and was purified by polyacrylamide gel electrophoresis (PAGE). Actin-as-250 was mixed with actin-s-140-m6A or actin-s-140-A at a molecular ratio 5:1 and coprecipitated. RNA pellets were resuspended in hybridization buffer, aliquoted, and incubated for 0.5, 2 and 4 h for sense–antisense annealing and duplex formation. After incubation, unhybridized RNA were digested by RNases A and T1. RNA samples were precipitated and then separated on a 5% acrylamide denaturing gel (National diagnostics). The gel was exposed to a storage phosphor screen and scanned by FujiFilm FLA-5100 imager.
6
Enzymatic Cleavage of DNA Adducts
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DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB). Incubations were performed at 37 °C for 8 h in NEBuffer 1 (hAAG and hAPE1) or ThermoPol Reaction Buffer (EcoFpg). DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol-precipitated as described above. Samples were resuspended in 10 μL of 90% formamide and heated at 50 °C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labeled DNA was visualized using FLA-5100 imager and analyzed using Multigauge software (Fujifilm).
7
2D-TLC Analysis of Labeled RNA
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