To ligate Illumina DIY adapters (as in Section 2.3.6 , described above), end-repair and A-tailing reactions were carried out in the same microtube but at a lower temperature. The following reagents were mixed in a new microtube: 10 μL of cleaned amplicons from 1st PCR, 5 μL of ligase buffer (B302 Sybenzyme), 5 μL of adapter mix, 0.5 μL of T4 DNA ligase (E330 Sybenzyme, Novosibirsk, Russia), 0.5 μL of 5′ deadenylase (M0331 NEB, Ipswich, MA, USA), 1 μL of 10 mM ATP (R0441 ThermoFisher, Waltham, MA, USA), 1 μL of Klenow exo- (m0212L NEB, Ipswich, MA, USA), 1 μL of dATP (R0141 ThermoFisher, Waltham, MA, USA), 2 μL of T4 PNK (EK0032 ThermoFisher, Waltham, MA, USA), 12 μL of 50% PEG-8000 solution, and 12 μL of MQ water. The mix was incubated at 37 °C for 40 min: 10 °C for 10 s, 30 °C for 30 s (100 cycles).
R0441
Manufactured by Thermo Fisher Scientific
Sourced in United States
The R0441 is a laboratory centrifuge designed for general-purpose applications. It is a compact and efficient device used to separate different components of a liquid mixture based on their relative densities. The centrifuge can accommodate various sample containers and operates at variable speeds to achieve the desired separation performance.
Automatically generated - may contain errors
Lab products found in correlation
R0461, by Thermo Fisher Scientific (2 mentions)
5 deadenylase, by New England Biolabs (1 mentions)
Klenow exo, by New England Biolabs (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
Detectx direct 2 3 cyclic gamp enzyme immunoassay kit, by Arbor Assays (1 mentions)
E 304, by R&D Systems (1 mentions)
Ubch7 ube2l3, by R&D Systems (1 mentions)
U 201, by R&D Systems (1 mentions)
γh2ax antibody, by Merck Group (1 mentions)
7 protocols using r0441
1
Illumina DIY Adapter Ligation Protocol
2
In Vitro Cyclic GAMP Assay
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0.8 μg of recombinant full-length human cGAS (Cayman, #22810) was used per single reaction in a 200 μl volume with 80 mM Tris–HCl (pH 7.5), 200 mM NaCl, 20 μM ZnCl2, 20 mM MgCl2, 0.25 mM GTP (Thermo Fisher Scientific #R0441) and 0.25 mM ATP (Thermo Fisher Scientific #R0461), with 20 μg of ISD70 (freshly annealed at 1 μg/μl in PBS), and 2 μl C2-Mut1/A151 diluted in TE buffer (to get 0.5, 2 or 10 μM ODN concentration in 200 μl). After 40 min at 37°C, 2.5 mM EDTA was added to each tube to stop the enzymatic reaction and the samples were either snap frozen and stored at –80°C or directly processed for cGAMP ELISA. cGAMP levels were measured with the DetectX Direct 2′,3′-Cyclic GAMP Enzyme Immunoassay Kit (Arbor Assays), according to the manufacturer's protocol. Briefly, 50 μl of in vitro reactions were added per well to the kit microplate with 50 μl Assay buffer, 25 μl conjugate, and 25 μl Antibody per well, prior to a minimum of 2 h incubation. The standards were prepared with serial-dilution in Assay buffer. Quantification of cGAMP levels was performed on a Fluostar OPTIMA (BMG LABTECH) plate-reader at 450 nm.
3
Modulating Retinal Cell Survival
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BMDMs were treated or not with 50 ng/mL lipopolysaccharide (LPS) (Escherichia coli 10004557; Thermos Fisher) for 24 h and then 1 mM of adenosine triphosphate (ATP; R0441; Thermo Fisher Scientific) was added for 30 min. BMDMs were washed twice with DMEM supplemented with 0.2% BSA (800-095-EG; Wisent). BMDMs were cultured for 24 h and the supernatant served as conditioned media. Mouse retinas were prepared and placed on either 100,000 adherent BMDMs for 18 h at 37 °C in DMEM with 0.2% BSA or in BMDM-derived conditioned media in the absence or presence of rytvela (1 μM), Kineret (1.5 mg/mL), or an anti-IL-1β antibody (150 ng/mL, Abcam 9722). Doses of rytvela and Kineret were determined based on our previous study [49 (link)]. After 18 h, the explants were evaluated by TUNEL assay.
4
Polyadenylation Kinetics Assay
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Cleavage reactions were set up as described above. To test polyadenylation, PAP was added to the cleavage reaction at a final concentration of either 12.5 nM or 25 nM. 3′-dATP (Merck C9137) and/or ATP (Thermo Scientific R0441) were also included. The assays were run and analyzed as described above for cleavage-only assays.
5
In vitro ubiquitination of 4E-BP2
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In vitro ubiquitination assay was performed in 100 μL reaction mixture at 37°C for 2 h. The reaction mixture included 100 ng purified human recombinant 4E-BP2 WT and N99D/N102D, 100 ng purified human recombinant UBE1 (E1 enzyme, E-304, BostonBiochem), 500 ng UbcH7/UBE2L3 (E2 enzyme, E2-640, BostonBiochem), 10 μg ubiquitin (U-530, BostonBiochem), 2.5 μg purified human recombinant CUL4B (E3 enzyme, H00008450-P01, Novus Biologicals), 50 ng purified human recombinant DDB1 (ab114333, abcam), purified human recombinant Raptor (H00057521-P01, Novus Biologicals) in an ATP-regenerating system [50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 2 mM ATP (R0441, ThermoFisher Scientific) 10 mM creatine phosphate (10621714001, Merck), 3.5 U/mL creatine kinase (10127566001, Merck) and 0.6 U/mL inorganic pyrophosphatase (M0361S, New England Biolabs)], in the presence of 5 μM ubiquitin aldehyde (U-201, BostonBiochem) and 50 μM MG132. Proteins were dissolved in Laemmli buffer and resolved by SDS-PAGE.
6
EGFR Dephosphorylation by PTPN2
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EGFR recombinant protein with kinase domain was purchased (Sino Biological, catalog no. 10001-H20B2) for in vitro dephosphorylation. For the in vitro dephosphorylation assay, the 6xHis-FKBP12F36V-PTPN2 protein was dialyzed in reaction buffer [20 mM tris, 300 mM NaCl, 0.02% Triton X-100, 2 mM DTT, and 2 mM MnCl2 (pH 7.5)]. A 5 μl of EGFR (160 nM) and 5 μl of 6xHis-FKBP12F36V-PTPN2 (80 nM) are added to 84 μl of reaction buffer along with 5 μl of ATP solution (1 mM; Thermo Fisher Scientific, catalog no. R0441) in a clear 96-well plate. GePhos1 (1 μl, 400 nM), inactive GePhos1 (1 μl, 4 nM), or DMSO (1 μL) was added accordingly. The plate was then incubated at 37°C for 1 hour, and phosphate release was monitored by Malachite Green assay according to the manufacturer’s protocol (Enzo Life Sciences, catalog no.BML-AK111-0250). Released phosphate concentrations were calculated from the standard curve generated with the phosphate standard solution.
7
Nucleotide Supplementation and DNA Damage Response
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U2‐OS PRDX1‐depleted and control cells (shPRDX1 & shNTC) were seeded in black Cellcarrier‐96‐well plates at 2,000 cells/well and incubated overnight for attachment. The cells were treated for 72 h with fresh media supplemented with nucleotides, each at 100 μM (R0451, R0471, R0461, R0441, Thermo Scientific). Next, 1 μM etoposide or DMSO (negative control, drug solvent) was added for 3 h following which cells were washed with PBS, and incubated with fresh media supplemented with nucleotides (100 μM each). Plates were fixed for 15 min with formaldehyde 4% at time points 0, 2, and 4 h. Immunofluorescence was performed with primary γH2AX antibody (05‐636, Merck) and secondary Alexa‐555 (A‐21424). Nuclei were visualized with DAPI staining.
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