dutp solution  (Thermo Fisher)

Journal: Bio-protocol

Article Title: TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions

doi: 10.21769/BioProtoc.2502

Figure Lengend Snippet: Micrographs showing salt stress-treated rice root tissue subjected to TUNEL assay to assess in situ DNA fragmentation. Root tissue from 15-day old rice seedlings was subjected to salinity stress (200 mM NaCl) for 60 h followed by TUNEL assay as per the protocol described above. F: Fluorescein (green), PI: Propidium Iodide (red), DAPI: 4’,6-diamidino-2-phenylindole (blue). Samples were mounted on slides in a mountant (ProLong ® Gold Antifade with DAPI, Thermo Fisher Scientific) and the slides were examined under Nikon A1R (Nikon, Japan) confocal microscope using a 20x objective. NIS Elements AR software (Nikon, Japan) was used to acquire and process the images. DAPI and PI were used to stain DNA (both damaged and undamaged). Fluorescein fluorescence (green) is due to incorporation of fluorescein-12-dUTP during the TUNEL reaction and would correspond to the number of free DNA ends. Scale bars = 100 µm.

Article Snippet: Fluorescein-12-dUTP* (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0101). dATP* (Promega, catalog number: U1202).

Techniques: TUNEL Assay, In Situ, Microscopy, Software, Staining, Fluorescence

Journal: Journal of molecular biology

Article Title: Derepression of SaPIbov1 is independent of φNM1 type 2 dUTPase activity and is inhibited by dUTP and dUMP

doi: 10.1016/j.jmb.2017.04.001

Figure Lengend Snippet: Progress of dUTP hydrolysis of WT and mutant Dut NM1 detected by 1 H-NMR. For each spectral overlay, the black spectrum is for free dUTP, while the red, blue, and green spectra were obtained at the indicated time points following enzyme addition. 1 H signal assignments for dUTP (black) and dUMP (blue) are indicated at the bottom. Asterisks indicate signals of residual imidazole.

Article Snippet: dUMP and dUTP (ThermoFisher Scientific) were used as obtained.

Techniques: Mutagenesis, Nuclear Magnetic Resonance

Journal: Nucleic Acids Research

Article Title: RNA structure analysis assisted by capillary electrophoresis

doi:

Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

Article Snippet: The standard labeling reaction with Klenow fragment was performed in a 10–20 µl volume and contained: 5 µM gel-purified transcript, 20 µM oligodeoxynucleotide templates (ex1a47nt-fl 5′- CA GTCCAGGAAGTCTCA, ex1b64nt-fl 5′- TA GGACACTCAGTGCCC, ex1a-fl 5′- CA CTTTACCCAGAGC and ex2-fl 5′- CA CAGATGGGACAC) complementary to the 3′ end of the four RNA molecules to be labeled: Ex1a47nt, Ex1b64nt, Ex1a and Ex1a-2, respectively (non-complementary positions underlined), 20 µM labeled dUTP (PE Applied Biosystems), 50 mM Tris–HCl (pH 7.2), 10 mM MgSO4 , 0.1 mM DTT, 20 U RNazin and 10 U Klenow fragment (Promega).

Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

Journal: Nucleic Acids Research

Article Title: RNA structure analysis assisted by capillary electrophoresis

doi:

Figure Lengend Snippet: Structure analysis of two BRCA1 transcripts: Ex1a-2 and Ex1b. ( A ) Non-denaturing 6% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1b transcript treated as follows: lane 1, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 2, treated as above but with carrier RNA added to a final concentration of 8 µM. ( B ) CE in non-denaturing conditions of Ex1b transcript fluorescently labeled at its 3′ end with TdT and [R110]dUTP (shadowed peak); ROX-500 internal standard (gray line). ( C and D ) As described in (A) and (B) but for Ex1a-2 transcript.

Article Snippet: The standard labeling reaction with Klenow fragment was performed in a 10–20 µl volume and contained: 5 µM gel-purified transcript, 20 µM oligodeoxynucleotide templates (ex1a47nt-fl 5′- CA GTCCAGGAAGTCTCA, ex1b64nt-fl 5′- TA GGACACTCAGTGCCC, ex1a-fl 5′- CA CTTTACCCAGAGC and ex2-fl 5′- CA CAGATGGGACAC) complementary to the 3′ end of the four RNA molecules to be labeled: Ex1a47nt, Ex1b64nt, Ex1a and Ex1a-2, respectively (non-complementary positions underlined), 20 µM labeled dUTP (PE Applied Biosystems), 50 mM Tris–HCl (pH 7.2), 10 mM MgSO4 , 0.1 mM DTT, 20 U RNazin and 10 U Klenow fragment (Promega).

Techniques: Polyacrylamide Gel Electrophoresis, Concentration Assay, Labeling

Journal: Nucleic Acids Research

Article Title: RNA structure analysis assisted by capillary electrophoresis

doi:

Figure Lengend Snippet: Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

Article Snippet: The standard labeling reaction with Klenow fragment was performed in a 10–20 µl volume and contained: 5 µM gel-purified transcript, 20 µM oligodeoxynucleotide templates (ex1a47nt-fl 5′- CA GTCCAGGAAGTCTCA, ex1b64nt-fl 5′- TA GGACACTCAGTGCCC, ex1a-fl 5′- CA CTTTACCCAGAGC and ex2-fl 5′- CA CAGATGGGACAC) complementary to the 3′ end of the four RNA molecules to be labeled: Ex1a47nt, Ex1b64nt, Ex1a and Ex1a-2, respectively (non-complementary positions underlined), 20 µM labeled dUTP (PE Applied Biosystems), 50 mM Tris–HCl (pH 7.2), 10 mM MgSO4 , 0.1 mM DTT, 20 U RNazin and 10 U Klenow fragment (Promega).

Techniques: End Labeling, Labeling

Journal: Scientific Reports

Article Title: Novel molecular marker-assisted strategy for production of wheat–Leymus mollis chromosome addition lines

doi: 10.1038/s41598-018-34545-x

Figure Lengend Snippet: Identification of L. mollis chromosomes added to wheat using genomic in situ hybridization (GISH). ( A – N ), L. mollis chromosomes; double letters, disomic lines; single letters, monosomic lines; arrows point to the added chromosomes detected with fluorescein-12-dUTP (green).

Article Snippet: L. mollis genomic DNA was labeled with fluorescein-12-dUTP (Thermo Scientific) using Random Primers DNA Labeling System (Invitrogen).

Techniques: In Situ Hybridization