RMI2-null HCT-116 clones were seeded onto 6-well dishes at 300 cells in three ml of media per well in triplicate for each cell line. The next day the cells were exposed to either 2 mJ UV (254 nm) or mock treatment using a GS Gene Linker UV Chamber (Bio-Rad). Cells were then grown for six days and then rinsed in PBS, fixed in ice-cold methanol and stained in crystal violet solution. The 6-well dishes were imaged and colonies of at least 0.032 mm2 were counted using ImageJ v2.0.0.
Gs gene linker uv chamber
Manufactured by Bio-Rad
Sourced in United States
The GS Gene Linker UV Chamber is a laboratory instrument used for the controlled exposure of samples to ultraviolet (UV) light. It is designed to facilitate various applications that require precise UV irradiation, such as nucleic acid crosslinking and photochemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent nucleic acid detection module kit, by Thermo Fisher Scientific (3 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Hybri dot manifold, by Thermo Fisher Scientific (2 mentions)
Ccl 81, by American Type Culture Collection (2 mentions)
Prime a gene labeling system, by Promega (1 mentions)
Phosphorimaging screens, by Bio-Rad (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nanoject 2 injector, by Drummond (1 mentions)
Dig dna labelling and detection kit, by Roche (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Pierce biotin 3 end dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Lightshift chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Hybond n positively charged nylon membrane, by Cytiva (1 mentions)
North2south chemiluminescence hybridization and detection kit, by Thermo Fisher Scientific (1 mentions)
Uvp chemstudio plus imager, by Analytik Jena (1 mentions)
Nucleospin gel and pcr clean up kit, by Takara Bio (1 mentions)
Quanti blue solution, by InvivoGen (1 mentions)
38 protocols using gs gene linker uv chamber
1
Clonogenic Assay for UV Damage
2
Genome Integration of Hygromycin Resistance in P. indica
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To verify integration of the hygromycin resistance cassette into the nuclear genome of P. indica, Southern blot analysis was performed. Genomic DNA from 7-day-old cultures grown on CM medium was extracted; 10–20 μg of extracted DNA was digested overnight with SacI (NEB). The digested DNA was separated on 0.9% TAE agarose gel for 5 h at 80 V and blotted onto a nylon membrane (AmershamBiosciences Hybond-N+, GE Healthcare) over night. The DNA was UV cross-linked to the membrane in a GS GENE LINKER UV chamber (BIO-RAD) using an auto cross-linking program (C2, 50 mμ Joule). The labeling of the Hygromycin B probe was performed using the Prime-a-Gene® Labeling System according to the manufacturer’s instructions (Promega). Hybridization and washing steps were performed at 65°C. The membrane was exposed on phosphorimaging screens (Bio-Rad) and signals were detected using a molecular imager and the Quantity One software (Bio-Rad).
3
Flock House Virus Infection Protocol
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Flock House virus was a kind gift from Dr Annette Schneemann (Scripps Research Institute, La Jolla, CA). FHV stock titer was determined at 2.92E + 06 TCID50/mL using the method as in (Eleftherianos et al. 2011 (link)). UV virus treatment (24,000 mJ of UV light) was done as described by (Merkling et al. 2015b (link)) using a GS Gene linker UV Chamber (Bio-Rad) (Supplementary Figure S1). Flies of desired sex, age, and genotype were individually injected with 4.6 nL of either virus stock solution or control 10 mM Tris–HCl pH 7.5 solution under CO2 anesthesia using a Nanoject II injector (Drummond Scientific). Flies were let to recover from the injection for ∼1 h at room temperature and then were placed in a 22°C incubator. For survival experiments, flies were separated by sex and placed in groups of 10 per vial for each experimental treatment. The number of living flies was recorded every 24 h. For virus load determination by RT–qPCR, flies were separated by sex and frozen in groups of five flies per experimental treatment at 4-, 5-, 6-, and 7-day post-infection (dpi) prior RNA extraction.
4
Deferred Antagonism Assay for S. mutans
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The deferred antagonism assay was performed as previously described (8 (link)). Briefly, 8 μL of overnight cultures of UA159, B04Sm5 ΔmucD, ΔmucF, ΔmucG, ΔmucH, ΔmucI, mucG-C, mucH-C, or mucI-C was spotted onto BHI + 1% agar or BHI + 1% agar that was buffered to pH 7 with 1 M KH2PO4/K2HPO4, pH 7.5, and incubated overnight at 37°C under 5% CO2/95% air. The following day, the plates were sterilized using the sterilization setting (90 s) in a GS Gene Linker UV Chamber (Bio-Rad, Inc.). 500 μL of overnight cultures of S. sanguinis SK36, S. gordonii ATCC 10558, or S. mitis F0392 was added to 5 mL molten BHI + 0.75% agar that had been cooled to 40°C, and this was used to overlay the plates with the S. mutans colonies. The agar overlay was allowed to solidify at room temperature, and then the plates were incubated overnight at 37°C under 5%CO2/95% air. Zones of inhibition were measured the following day using a ruler.
5
Phage Induction and Probe Detection
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Bacteriophage were induced using Mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) as previously described [10 (link)]. Plaques were transferred onto nylon membranes using standard techniques [32 ] and DNA was cross-linked to the membrane (Amersham Biosciences, Little Chalfont, Bucks, England) using a GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were treated with 2 mg/mL Proteinase K (Roche Diagnostics, Mannheim, Germany). The hybridisation probe was obtained by PCR amplification of lukSF-PV using previously described primers [33 (link)]. PCR products were purified using the MoBio PCR Cleanup Kit. Probes were prepared using the DIG DNA Labelling and Detection Kit according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). Plaque hybridisation was performed as directed by the manufacturer (Boehringer Mannheim, Mannheim, Germany).
6
Apoptosis Induction in Cell Lines
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HeLa, HEK-293T and HCT116 cells were obtained from the Cell Resource Center of Peking Union Medical College (PUMC). HeLa and HEK-293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS); HCT116 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS. All of the cell lines were cultured in a 5% CO2 incubator at 37°C, and they were passaged every 2–3 days with 0.5 mg/ml trypsin (1∶250) and 0.53 mM ethylenediaminetetraacetic acid (EDTA). To induce apoptosis, HeLa cells were treated with 20 ng/ml TNFα, 50 ng/ml TRAIL or 0.2 μM Stauriprione for 12 hours. For ultraviolet light (UV) treatment, DMEM was displaced with PBS, and cells were exposed to 40 J/m2 UV irradiation in a GS Genelinker UV chamber (Bio-Rad). The cells were then maintained in DMEM and harvested at indicated times.
7
HCMV Inactivation by UV Exposure
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For FIG 5 , HCMV strain Towne (MOI=2 TCID50/cell) was placed on ice for 5 minutes and either mock-treated or treated with 125 mJ of UV light for 0, 15, 30, 45 or 60 seconds (Bio-Rad GS Gene Linker UV Chamber). The inoculum was placed on fibroblasts for 12 hours, lysates were gathered and a Western blot analysis was run as described above. For figure 4 B –D , HCMV strain Towne (MOI=2 TCID50/cell) was placed on ice for 5 minutes and either mock-treated or treated with 125 mJ of UV light for 60 seconds. Lysates were gathered at indicated time points and a Western blot analysis was run as described above.
8
Protein-DNA Binding Assay Protocol
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The upstream regions of the toxP, cbuA0029, and groEL genes were first selected for PCR amplification. PCR products (1 μg) of desired templates were 3′ end‐labeled using a Pierce biotin 3′ End DNA Labeling Kit (Thermo Scientific). The resulting probe reaction mixtures were electrophoresed on a 0.8% agarose gel for 30 min at 100 V and then gel purified with a NucleoSpin Gel and PCR Clean‐up kit (Takara Bio USA). The EMSA binding reaction, consisting of 2.5% glycerol, 5 mM MgCl2, 50 mM KCl, 1 nM biotin‐labeled DNA, and varying concentrations of either ToxP or AntitoxP/ToxP in 1X Binding Buffer (LightShift Chemiluminescence kit; Thermo Scientific), was assembled and incubated at room temperature for 30 min. A nondenaturing loading dye (0.25% bromophenol blue) was added, and the resulting RNA mixtures were resolved on a 10% polyacrylamide gel for 2 h at 100 V. DNA/protein complexes were transferred to a Hybond‐N+ positively charged nylon membrane (Amersham Pharmacia Biotech) using an electroblot transfer system (Bio‐Rad) and cross‐linked with short‐wave UV light in a GS gene linker UV chamber (Bio‐Rad). A North2South chemiluminescence hybridization and detection kit (Thermo Scientific) was used to detect resulting bands. The blot was imaged on a UVP ChemStudio PLUS Imager (Analytik Jena).
9
C. elegans Culturing and Sterilization
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C. elegans strains used in this study are listed in Supplementary Data 1 . Worms were grown on NGM plates with standard techniques at 20 °C47 (link). All assayed worms were at day 1 of adulthood unless otherwise noted. Some strains were provided by CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). OP50 colonies on an NGM plate were treated with the sterilisation programme in a GS Gene Linker UV Chamber (BIO-RAD) for 15 m to obtain UV-killed bacteria. The killed bacteria were confirmed by no growth after O/N incubation in LB.
We have complied with all relevant ethical regulations for animal testing and research. The study has been approved by the Ethics Committee of Shanghai Institute of Biochemistry and Cell Biology, CAS.
We have complied with all relevant ethical regulations for animal testing and research. The study has been approved by the Ethics Committee of Shanghai Institute of Biochemistry and Cell Biology, CAS.
10
Assessing Bioactive IL-1β in Conditioned Media
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HEK-Blue IL-1β cells (hkb-il1bv2, InvivoGen, San Diego, CA, USA) were seeded at 150 μL per 96-well plate overnight, then incubated with 50 μL UV-inactivated conditioned media for another day. UV inactivation was done by using the GS Gene Linker UV Chamber (Bio-Rad, Hercules, CA, USA) following the program setting of sterilization application. For the confirmation of virus inactivation in the experiments, a parallel experiment was done side by side stained with ZIKV viral protein to ensure no virus was alive. HEK-Blue IL-1β cells sense bioactive IL-1β in conditioned media and then activate nuclear factor κB (NF-κB)/AP-1, leading to the production of secreted embryonic alkaline phosphatase (SEAP). The cell culture supernatant was transferred to a flat-bottom 96-well plate and mixed with 150 μL/well QUANTI-Blue Solution, a SEAP detection medium (rep-qbs, InvivoGen). After incubation, the bioactive IL-1β was represented by measuring the SEAP levels by spectrophotometry at 650 nm.
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