Structured Review

Thermo Fisher mgcl2
Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 10037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Amplification:

Article Title: Molecular identification of Lodoicea maldivica (coco de mer) seeds
Article Snippet: .. PCR for PRK gene The PRK gene was amplified with 50 ng DNA template, 1.25 μL of 5 mM dNTPs, 2.0 μL of 25 mM MgCl2 , 2.5 μL of 10× PCR buffer without MgCl2 (Applied Biosystems, USA), 1.5 μL of 10 μM specific primers (Table ), 1.25 μL of 40 ng/μL bovine serum albumin (BSA), 0.5 μL of dimethyl sulfoxide (DMSO) and 1 U of AmpliTaq Gold polymerase (Applied Biosystems, USA). ..

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: .. Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer. .. Both HVI (15975-16501) and HVII (15-410) regions were amplified in a single tube using a set of MID-tagged fusion primers.

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: Subsequent PCR amplification of the RT reaction using the following primer pair was performed as follows: TREM-1 upstream primer: 5′-AGG GGC CAC ACC AAC CTT CTG-3′, TREM-1 downstream primer: 5′-AGT GCC TGC CTC AAT GTC TCC A-3′, annealing temperature: 65°C; product length: 364 base pairs (bp). .. All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany).

Article Title: Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9?-Hydroxylase Enzymes
Article Snippet: The chimeric genes kshA1A5loop , kshA1A5TG , and kshA5A1loop ( ) were made via ligase-independent cloning (LIC) ( ) using pKsh814 or pA5rho5 (see Table S1 in the supplemental material) as the DNA template for PCR amplification. .. The PCR mixtures consisted of Tris HCl (10 mM, pH 8.0), 1× polymerase buffer with 1.5 mM MgCl2 , MgCl2 (2.5 mM), deoxyribonucleotide triphosphates (0.2 mM), dimethyl sulfoxide (2%), primers (10 ng μl−1 ), High Fidelity DNA polymerase (0.5 units [25 μl−1 ]; Fermentas), and 1 pg DNA template under the following conditions: 5 min at 95°C and 30 cycles of 1 min at 95°C, 45 s at 68°C, and 4 min at 72°C, followed by 5 min at 72°C.

High Throughput Screening Assay:

Article Title: Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae) 1
Article Snippet: PCR amplifications were performed in 25-μL reaction mixtures with 50 ng of template DNA, 0.2 μM of each forward and reverse primers, 200 μM dNTPs, 2.5 mM MgCl2 , 1× PCR buffer, and 0.5 U Taq DNA polymerase in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Carlsbad, California, USA). .. Length polymorphism was viewed with ethidium bromide in 8% polyacrylamide gels run in a Mega-Gel high-throughput electrophoresis system for 5 h at 250 V (C.B.S.

Polymerase Chain Reaction:

Article Title: Molecular identification of Lodoicea maldivica (coco de mer) seeds
Article Snippet: .. PCR for PRK gene The PRK gene was amplified with 50 ng DNA template, 1.25 μL of 5 mM dNTPs, 2.0 μL of 25 mM MgCl2 , 2.5 μL of 10× PCR buffer without MgCl2 (Applied Biosystems, USA), 1.5 μL of 10 μM specific primers (Table ), 1.25 μL of 40 ng/μL bovine serum albumin (BSA), 0.5 μL of dimethyl sulfoxide (DMSO) and 1 U of AmpliTaq Gold polymerase (Applied Biosystems, USA). ..

Article Title: Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae) 1
Article Snippet: .. PCR amplifications were performed in 25-μL reaction mixtures with 50 ng of template DNA, 0.2 μM of each forward and reverse primers, 200 μM dNTPs, 2.5 mM MgCl2 , 1× PCR buffer, and 0.5 U Taq DNA polymerase in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Carlsbad, California, USA). .. The PCR profile included an initial denaturation at 95°C for 3 min followed by 35 cycles of 95°C for 1 min, 51–62°C for 50 s (according to the primer’s annealing temperature), 72°C for 1 min, and a final extension at 72°C for 10 min.

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: .. Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer. .. Both HVI (15975-16501) and HVII (15-410) regions were amplified in a single tube using a set of MID-tagged fusion primers.

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: .. All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany). .. PCR conditions were as follows: initially at 94°C for 2 min, 94°C for 30 s, annealing at the primer-specific temperature for 30 s, 30 s at 72°C for 40 cycles and finally at 72°C for 5 min.

Article Title: Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9?-Hydroxylase Enzymes
Article Snippet: .. The PCR mixtures consisted of Tris HCl (10 mM, pH 8.0), 1× polymerase buffer with 1.5 mM MgCl2 , MgCl2 (2.5 mM), deoxyribonucleotide triphosphates (0.2 mM), dimethyl sulfoxide (2%), primers (10 ng μl−1 ), High Fidelity DNA polymerase (0.5 units [25 μl−1 ]; Fermentas), and 1 pg DNA template under the following conditions: 5 min at 95°C and 30 cycles of 1 min at 95°C, 45 s at 68°C, and 4 min at 72°C, followed by 5 min at 72°C. .. Site-directed mutagenesis (QuikChange protocol; see above) was used to generate kshA1D242W and kshA5W248D ( ).

Quantitative RT-PCR:

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: Total RNA was used for reverse transcription real-time PCR (RT-qPCR) and total protein for Western blotting, as previously described ( ). .. Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific).

Electrophoresis:

Article Title: Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae) 1
Article Snippet: PCR amplifications were performed in 25-μL reaction mixtures with 50 ng of template DNA, 0.2 μM of each forward and reverse primers, 200 μM dNTPs, 2.5 mM MgCl2 , 1× PCR buffer, and 0.5 U Taq DNA polymerase in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Carlsbad, California, USA). .. Length polymorphism was viewed with ethidium bromide in 8% polyacrylamide gels run in a Mega-Gel high-throughput electrophoresis system for 5 h at 250 V (C.B.S.

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer. .. PCR products were analyzed on 1.5% agarose gels in 1X Tris-Acetate-EDTA electrophoresis buffer (TAE) stained with 0.5 µg/mL ethidium bromide to confirm successful amplification of the target regions and to assess primer dimer and PCR specificity.

Knock-In:

Article Title: Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells
Article Snippet: Cre+;Cdc42FL-MA9 cell line: Murine AML cells derived from knock-in of the MLL-AF9 (MA9; also known as KMT2A-MLLT3) oncogene into the ROSA26CreERt2;Cdc42FL/FL background. .. Phosphate-buffered saline (PBS) with 1 mM CaCl2 and MgCl2 (Gibco).

Incubation:

Article Title: Enhancing the effects of chemotherapy by combined macrophage-mediated photothermal therapy (PTT) and photochemical internalization (PCI)
Article Snippet: .. The Ma were incubated for 24 h at 37 °C, rinsed three times with Hanks’ Balanced Salt Solution with calcium chloride and magnesium chloride (HBSS, Gibco, Carlsbad, CA) to wash away the excess of noningested nanoshells. ..

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: .. For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen). ..

Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
Article Snippet: .. Second strand cDNA was prepared by incubating 0.1 µg/µl total RNA in 50 µl of 50 mM Tris–HCl pH 8.3, 3 mM MgCl2 , 75 mM KCl, 10 mM DTT, 1 µM random hexamer primers (Gibco BRL), 0.8 U/µl HPRI ribonuclease inhibitor (Amersham Bioscience) and 3 U/µl M-MLV reverse transcriptase (USB) for 30 s at 50°C, followed by 1 h at 37°C and finally 80°C for 10 min. An aliquot of 5 µl of second strand reaction mix, containing 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 0.25 µg BSA, 5 µM random primers and 5 U Klenow fragment (Amersham Bioscience) was added to 20 µl of the first strand reaction and incubated at 37°C for 1 h, followed by 65°C for 15 min. .. All padlock probes were evaluated using Oligo 6.6 software (Molecular Biology Insights Inc.) to avoid secondary structure that might interfere with probe function.

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5). .. For m6 A and m6 Am in polyadenylated RNA, 200 ng polyadenylated RNA purified from HEK293T cells was incubated with 2 M FTO purified in mammalian cells (High concentration) or 0.2 M FTO (low concentration) in the above reaction buffer for 3 hours and then quenched by the addition of 5 mM of EDTA, respectively.

Activity Assay:

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific). .. The same protocol was used to assess senescence-associated β-galactosidase (SA-β-Gal) activity, but using a staining solution at a pH of 6.

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: Paragraph title: Biochemistry assay of FTO activity in vitro ... Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5).

Modification:

Article Title: Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells
Article Snippet: Phosphate-buffered saline (PBS) with 1 mM CaCl2 and MgCl2 (Gibco). .. Medium: Iscove’s modified Dulbecco’s medium with L-gluta-mine and HEPES (IMDM; Corning cellgro) supplemented with 20% heat-inactivated FBS, 1% Pen-Strep, and cytokine mix as noted below ( item 6 ).

Article Title: Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae) 1
Article Snippet: Genomic DNA of 16 pea genotypes including widely grown cultivars and plant introduction lines (i.e., Shawnee, Melrose, Medora, Lifter, Radley, PI 179449, Green Arrow, Frolic, A778-26-6, Sparkle, JI73, Bohatyr, ICI12043, PI 240515, PI 103709, PI 169603) was extracted from leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) extraction protocol ( ). .. PCR amplifications were performed in 25-μL reaction mixtures with 50 ng of template DNA, 0.2 μM of each forward and reverse primers, 200 μM dNTPs, 2.5 mM MgCl2 , 1× PCR buffer, and 0.5 U Taq DNA polymerase in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Carlsbad, California, USA).

Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction
Article Snippet: Human embryonic kidney 293T (HEK293T), HeLa, and MCF7/LC3-EGFP cells were grown in Dulbecco’s modified eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 4,500 mg/liter of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin (Sigma-Aldrich), and 1 mM l -alanyl- l -glutamine (Sigma-Aldrich) at 37°C in 5% CO2 . .. For amino acid starvation to induce of autophagy in MCF7/LC3-EGFP cells, Hanks’ balanced salt solution (HBSS) with CaCl2 and MgCl2 was used (Gibco, Carlsbad, CA, USA).

Western Blot:

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: Total RNA was used for reverse transcription real-time PCR (RT-qPCR) and total protein for Western blotting, as previously described ( ). .. Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific).

Derivative Assay:

Article Title: Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells
Article Snippet: Cre+;Cdc42FL-MA9 cell line: Murine AML cells derived from knock-in of the MLL-AF9 (MA9; also known as KMT2A-MLLT3) oncogene into the ROSA26CreERt2;Cdc42FL/FL background. .. Phosphate-buffered saline (PBS) with 1 mM CaCl2 and MgCl2 (Gibco).

Electron Microscopy:

Article Title: Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens
Article Snippet: 179337) Paraformaldehyde (PFA) (Electron microscopy Sciences, cat.no. .. 13800) Fluoromount G (Southern Biotech, cat.no.17984-25) Dulbecco’s PBS with CaCl2 and MgCl2 (Life Technologies, cat.no.

Concentration Assay:

Article Title: Enhancing the effects of chemotherapy by combined macrophage-mediated photothermal therapy (PTT) and photochemical internalization (PCI)
Article Snippet: The Ma were incubated for 24 h at 37 °C, rinsed three times with Hanks’ Balanced Salt Solution with calcium chloride and magnesium chloride (HBSS, Gibco, Carlsbad, CA) to wash away the excess of noningested nanoshells. .. The concentration of nanoshells in macrophages was studied using a UV-Vis-NIR spectrophotometer (Varian UV-Vis-NIR spectrophotometer Cary 6000i, Varian, USA).

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: .. All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany). .. PCR conditions were as follows: initially at 94°C for 2 min, 94°C for 30 s, annealing at the primer-specific temperature for 30 s, 30 s at 72°C for 40 cycles and finally at 72°C for 5 min.

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5). .. For m6 A and m6 Am in polyadenylated RNA, 200 ng polyadenylated RNA purified from HEK293T cells was incubated with 2 M FTO purified in mammalian cells (High concentration) or 0.2 M FTO (low concentration) in the above reaction buffer for 3 hours and then quenched by the addition of 5 mM of EDTA, respectively.

Protease Inhibitor:

Article Title: Excised linear introns regulate growth in yeast
Article Snippet: .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]). .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]).

Cell Culture:

Article Title: Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells
Article Snippet: Paragraph title: 2.1. Cell Culture ... Phosphate-buffered saline (PBS) with 1 mM CaCl2 and MgCl2 (Gibco).

Article Title: Enhancing the effects of chemotherapy by combined macrophage-mediated photothermal therapy (PTT) and photochemical internalization (PCI)
Article Snippet: NR8383 rat alveolar Ma were seeded in 35-mm cell culture dishes at 1 × 106 Ma in 2 ml of culture medium. .. The Ma were incubated for 24 h at 37 °C, rinsed three times with Hanks’ Balanced Salt Solution with calcium chloride and magnesium chloride (HBSS, Gibco, Carlsbad, CA) to wash away the excess of noningested nanoshells.

Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction
Article Snippet: Paragraph title: Cell culture. ... For amino acid starvation to induce of autophagy in MCF7/LC3-EGFP cells, Hanks’ balanced salt solution (HBSS) with CaCl2 and MgCl2 was used (Gibco, Carlsbad, CA, USA).

Sedimentation:

Article Title: Excised linear introns regulate growth in yeast
Article Snippet: Paragraph title: Sedimentation velocity ... Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]).

Generated:

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen). .. The capacity of cross-linked UvrB and generated mutants to associate with UvrA was assessed by non-denaturing PAGE using the Novex® Bis-Tris Gel System (Invitrogen).

Ligase Independent Cloning:

Article Title: Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9?-Hydroxylase Enzymes
Article Snippet: The chimeric genes kshA1A5loop , kshA1A5TG , and kshA5A1loop ( ) were made via ligase-independent cloning (LIC) ( ) using pKsh814 or pA5rho5 (see Table S1 in the supplemental material) as the DNA template for PCR amplification. .. The PCR mixtures consisted of Tris HCl (10 mM, pH 8.0), 1× polymerase buffer with 1.5 mM MgCl2 , MgCl2 (2.5 mM), deoxyribonucleotide triphosphates (0.2 mM), dimethyl sulfoxide (2%), primers (10 ng μl−1 ), High Fidelity DNA polymerase (0.5 units [25 μl−1 ]; Fermentas), and 1 pg DNA template under the following conditions: 5 min at 95°C and 30 cycles of 1 min at 95°C, 45 s at 68°C, and 4 min at 72°C, followed by 5 min at 72°C.

Sequencing:

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: Unique 10 base MID tags are used as sample identifiers in order to pool and sequence multiple samples in a single 454 sequencing run with each sample being “tagged” or “barcoded” with a different MID tag. .. Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer.

Binding Assay:

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: .. For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen). ..

Immunofluorescence:

Article Title: Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens
Article Snippet: Paragraph title: Immunofluorescence staining of malaria-infected MPCCs ... 13800) Fluoromount G (Southern Biotech, cat.no.17984-25) Dulbecco’s PBS with CaCl2 and MgCl2 (Life Technologies, cat.no.

Nucleic Acid Electrophoresis:

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany). .. Ten μl of each PCR reaction was size-fractionated by gel electrophoresis in a 2% agarose gel and analysed.

Fluorescence:

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen). .. All gels were visualized using a UV transilluminator to detect the intrinsic fluorescence of the fluorescein moiety.

Mutagenesis:

Article Title: Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9?-Hydroxylase Enzymes
Article Snippet: The PCR mixtures consisted of Tris HCl (10 mM, pH 8.0), 1× polymerase buffer with 1.5 mM MgCl2 , MgCl2 (2.5 mM), deoxyribonucleotide triphosphates (0.2 mM), dimethyl sulfoxide (2%), primers (10 ng μl−1 ), High Fidelity DNA polymerase (0.5 units [25 μl−1 ]; Fermentas), and 1 pg DNA template under the following conditions: 5 min at 95°C and 30 cycles of 1 min at 95°C, 45 s at 68°C, and 4 min at 72°C, followed by 5 min at 72°C. .. Site-directed mutagenesis (QuikChange protocol; see above) was used to generate kshA1D242W and kshA5W248D ( ).

Electrophoretic Mobility Shift Assay:

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: Paragraph title: Gel-shift assays ... For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen).

Mouse Assay:

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: The mice were sacrificed and 3 lobes of the right lung were quickly removed and immediately snap-frozen in liquid nitrogen and then stored at –80°C for biological measurements. .. Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: Paragraph title: Measurement of TREM-1 by semiquantitative RT–PCR ... All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany).

Construct:

Article Title: Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9?-Hydroxylase Enzymes
Article Snippet: The chimeric genes kshA1 A5 β201 -210 and kshA1 A5 β230 -238 (pKSH814 as template) and kshA5 A1 β207 -216 and kshA5 A1 β236 -244 (pA5rho5 as template) were constructed by PCR techniques ( ). .. The PCR mixtures consisted of Tris HCl (10 mM, pH 8.0), 1× polymerase buffer with 1.5 mM MgCl2 , MgCl2 (2.5 mM), deoxyribonucleotide triphosphates (0.2 mM), dimethyl sulfoxide (2%), primers (10 ng μl−1 ), High Fidelity DNA polymerase (0.5 units [25 μl−1 ]; Fermentas), and 1 pg DNA template under the following conditions: 5 min at 95°C and 30 cycles of 1 min at 95°C, 45 s at 68°C, and 4 min at 72°C, followed by 5 min at 72°C.

Polyacrylamide Gel Electrophoresis:

Article Title: Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes
Article Snippet: .. For the UvrB self-loading assays, 2.7 µM UvrB (or UvrB2 X) was incubated with 200 nM G10 in binding buffer (20 mM Tris–HCl (pH 7.6), 50 mM NaCl, 4% Ficoll, 10 mM MgCl2 and 10 mM ATP) for 30 min at 25°C before loading onto a pre-cast 6% PAGE gel (Invitrogen). ..

Staining:

Article Title: Molecular identification of Lodoicea maldivica (coco de mer) seeds
Article Snippet: PCR for PRK gene The PRK gene was amplified with 50 ng DNA template, 1.25 μL of 5 mM dNTPs, 2.0 μL of 25 mM MgCl2 , 2.5 μL of 10× PCR buffer without MgCl2 (Applied Biosystems, USA), 1.5 μL of 10 μM specific primers (Table ), 1.25 μL of 40 ng/μL bovine serum albumin (BSA), 0.5 μL of dimethyl sulfoxide (DMSO) and 1 U of AmpliTaq Gold polymerase (Applied Biosystems, USA). .. The amplification products were electrophoresed on a 1% agarose gel, stained with ethidium bromide and observed under UV illumination.

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: .. Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific). .. The same protocol was used to assess senescence-associated β-galactosidase (SA-β-Gal) activity, but using a staining solution at a pH of 6.

Article Title: Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens
Article Snippet: Paragraph title: Immunofluorescence staining of malaria-infected MPCCs ... 13800) Fluoromount G (Southern Biotech, cat.no.17984-25) Dulbecco’s PBS with CaCl2 and MgCl2 (Life Technologies, cat.no.

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer. .. PCR products were analyzed on 1.5% agarose gels in 1X Tris-Acetate-EDTA electrophoresis buffer (TAE) stained with 0.5 µg/mL ethidium bromide to confirm successful amplification of the target regions and to assess primer dimer and PCR specificity.

Chloramphenicol Acetyltransferase Assay:

Article Title: Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens
Article Snippet: .. 13800) Fluoromount G (Southern Biotech, cat.no.17984-25) Dulbecco’s PBS with CaCl2 and MgCl2 (Life Technologies, cat.no. .. 14040) Bovine serum albumin (BSA) (Sigma-Aldrich, cat.no.

Purification:

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5). .. For m6 A and m6 Am in polyadenylated RNA, 200 ng polyadenylated RNA purified from HEK293T cells was incubated with 2 M FTO purified in mammalian cells (High concentration) or 0.2 M FTO (low concentration) in the above reaction buffer for 3 hours and then quenched by the addition of 5 mM of EDTA, respectively.

Real-time Polymerase Chain Reaction:

Article Title: The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice
Article Snippet: Total RNA was used for reverse transcription real-time PCR (RT-qPCR) and total protein for Western blotting, as previously described ( ). .. Then, the lungs were washed with PBS and stained in a titrated pH 7.4 solution containing 40 mM citric acid, 150 mM NaCl, 2 mM MgCl2 , 5 mM potassium ferrocyanide, and 1 mg/mL X-Gal (Thermo Fisher Scientific).

Agarose Gel Electrophoresis:

Article Title: Molecular identification of Lodoicea maldivica (coco de mer) seeds
Article Snippet: PCR for PRK gene The PRK gene was amplified with 50 ng DNA template, 1.25 μL of 5 mM dNTPs, 2.0 μL of 25 mM MgCl2 , 2.5 μL of 10× PCR buffer without MgCl2 (Applied Biosystems, USA), 1.5 μL of 10 μM specific primers (Table ), 1.25 μL of 40 ng/μL bovine serum albumin (BSA), 0.5 μL of dimethyl sulfoxide (DMSO) and 1 U of AmpliTaq Gold polymerase (Applied Biosystems, USA). .. The amplification products were electrophoresed on a 1% agarose gel, stained with ethidium bromide and observed under UV illumination.

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany). .. Ten μl of each PCR reaction was size-fractionated by gel electrophoresis in a 2% agarose gel and analysed.

In Vitro:

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: Paragraph title: Biochemistry assay of FTO activity in vitro ... Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5).

Random Hexamer Labeling:

Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
Article Snippet: .. Second strand cDNA was prepared by incubating 0.1 µg/µl total RNA in 50 µl of 50 mM Tris–HCl pH 8.3, 3 mM MgCl2 , 75 mM KCl, 10 mM DTT, 1 µM random hexamer primers (Gibco BRL), 0.8 U/µl HPRI ribonuclease inhibitor (Amersham Bioscience) and 3 U/µl M-MLV reverse transcriptase (USB) for 30 s at 50°C, followed by 1 h at 37°C and finally 80°C for 10 min. An aliquot of 5 µl of second strand reaction mix, containing 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 0.25 µg BSA, 5 µM random primers and 5 U Klenow fragment (Amersham Bioscience) was added to 20 µl of the first strand reaction and incubated at 37°C for 1 h, followed by 65°C for 15 min. .. All padlock probes were evaluated using Oligo 6.6 software (Molecular Biology Insights Inc.) to avoid secondary structure that might interfere with probe function.

Spectrophotometry:

Article Title: Enhancing the effects of chemotherapy by combined macrophage-mediated photothermal therapy (PTT) and photochemical internalization (PCI)
Article Snippet: The Ma were incubated for 24 h at 37 °C, rinsed three times with Hanks’ Balanced Salt Solution with calcium chloride and magnesium chloride (HBSS, Gibco, Carlsbad, CA) to wash away the excess of noningested nanoshells. .. The concentration of nanoshells in macrophages was studied using a UV-Vis-NIR spectrophotometer (Varian UV-Vis-NIR spectrophotometer Cary 6000i, Varian, USA).

Activation Assay:

Article Title: Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions
Article Snippet: Each sample was amplified in a 50 µL PCR reaction containing 1.2X GeneAmp® PCR Buffer II without MgCl2 (Life Technologies, Foster City, CA, USA), 2.4 mM MgCl2 , 0.2 mM each dNTP (Roche Life Sciences, Indianapolis, IN, USA), 0.25 U/µL EagleTaq DNA Polymerase (Roche Custom Biotech, Indianapolis, IN, USA), and 0.3 µM each primer. .. All reactions were performed in GeneAmp® PCR Systems 9600 thermal cycler (Applied Biosystems, Carlsbad, CA, USA) using the following cycling conditions: a 14-minute 94°C activation cycle; 34 cycles of amplification at 94°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; and a final 10-minute 72°C extension cycle.

Demethylation Activity Assay:

Article Title: Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in Cell Nucleus and Cytoplasm
Article Snippet: .. Similar to a previous report , the demethylation activity assay was performed in standard 20 μl of reaction buffer containing KCl (100 mM), MgCl2 (2 mM), SUPERNase In (0.2 U/μl, life technology), L-ascorbic acid (2 mM), α-ketoglutarate (300 M), (NH4 )2 Fe(SO4 )2 ·6H2 O (150 μM), and 50 mM of HEPES buffer (pH 6.5). .. For m6 A and m6 Am in polyadenylated RNA, 200 ng polyadenylated RNA purified from HEK293T cells was incubated with 2 M FTO purified in mammalian cells (High concentration) or 0.2 M FTO (low concentration) in the above reaction buffer for 3 hours and then quenched by the addition of 5 mM of EDTA, respectively.

CTG Assay:

Article Title: Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection
Article Snippet: Subsequent PCR amplification of the RT reaction using the following primer pair was performed as follows: TREM-1 upstream primer: 5′-AGG GGC CAC ACC AAC CTT CTG-3′, TREM-1 downstream primer: 5′-AGT GCC TGC CTC AAT GTC TCC A-3′, annealing temperature: 65°C; product length: 364 base pairs (bp). .. All PCR reactions were performed in the GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, CA, USA) in a final volume of 25 μl containing 2 μl cDNA, 2·5 μl 10× buffer without MgCl2 , 1·25 μl of 50 mM MgCl2 (final concentration 2·5 mM), 5 μl of deoxynucleotide triphosphate (dNTP)-mix, 1 mM of each nucleotide (final concentration 0·2 mM each) (Invitrogen, Karlsruhe, Germany), 10 pmol of each primer (final concentration 0·4 μM each), 0·1 μl of Taq DNA polymerase (5 U/μl) (Invitrogen, Karlsruhe, Germany).

Lysis:

Article Title: Excised linear introns regulate growth in yeast
Article Snippet: .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]). .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]).

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  • 90
    Thermo Fisher bodipy fl atp
    Single-molecule binding assay of AK and <t>ATP.</t> (A) Typical fluorescence trajectory of <t>BODIPY</t> FL-ATP associating and dissociating with AK. Association and dissociation are indicated by red and green bands, respectively. (B) Evolution of the mean intensity of BODIPY FL-ATP with time. By fitting with single exponential decay, we obtain the bleaching rate constant k 2 = 0.22 ±0.01 s −1 . (C,D) Histograms of t off and t on event durations. By fitting with distribution shown in Equation (1) and (2), we obtain the association rate constant k 1 = 1.5 ± 0.2 μM −1 s −1 and the dissociation rate constant k −1 = 6.9 ± 0.7 s −1 .
    Bodipy Fl Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Single-molecule binding assay of AK and <t>ATP.</t> (A) Typical fluorescence trajectory of <t>BODIPY</t> FL-ATP associating and dissociating with AK. Association and dissociation are indicated by red and green bands, respectively. (B) Evolution of the mean intensity of BODIPY FL-ATP with time. By fitting with single exponential decay, we obtain the bleaching rate constant k 2 = 0.22 ±0.01 s −1 . (C,D) Histograms of t off and t on event durations. By fitting with distribution shown in Equation (1) and (2), we obtain the association rate constant k 1 = 1.5 ± 0.2 μM −1 s −1 and the dissociation rate constant k −1 = 6.9 ± 0.7 s −1 .
    Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 10037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taq buffer
    Single-molecule binding assay of AK and <t>ATP.</t> (A) Typical fluorescence trajectory of <t>BODIPY</t> FL-ATP associating and dissociating with AK. Association and dissociation are indicated by red and green bands, respectively. (B) Evolution of the mean intensity of BODIPY FL-ATP with time. By fitting with single exponential decay, we obtain the bleaching rate constant k 2 = 0.22 ±0.01 s −1 . (C,D) Histograms of t off and t on event durations. By fitting with distribution shown in Equation (1) and (2), we obtain the association rate constant k 1 = 1.5 ± 0.2 μM −1 s −1 and the dissociation rate constant k −1 = 6.9 ± 0.7 s −1 .
    Taq Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Single-molecule binding assay of AK and ATP. (A) Typical fluorescence trajectory of BODIPY FL-ATP associating and dissociating with AK. Association and dissociation are indicated by red and green bands, respectively. (B) Evolution of the mean intensity of BODIPY FL-ATP with time. By fitting with single exponential decay, we obtain the bleaching rate constant k 2 = 0.22 ±0.01 s −1 . (C,D) Histograms of t off and t on event durations. By fitting with distribution shown in Equation (1) and (2), we obtain the association rate constant k 1 = 1.5 ± 0.2 μM −1 s −1 and the dissociation rate constant k −1 = 6.9 ± 0.7 s −1 .

    Journal: Frontiers in Plant Science

    Article Title: Single-Molecule Fluorescence Methods to Study Plant Hormone Signal Transduction Pathways

    doi: 10.3389/fpls.2017.01888

    Figure Lengend Snippet: Single-molecule binding assay of AK and ATP. (A) Typical fluorescence trajectory of BODIPY FL-ATP associating and dissociating with AK. Association and dissociation are indicated by red and green bands, respectively. (B) Evolution of the mean intensity of BODIPY FL-ATP with time. By fitting with single exponential decay, we obtain the bleaching rate constant k 2 = 0.22 ±0.01 s −1 . (C,D) Histograms of t off and t on event durations. By fitting with distribution shown in Equation (1) and (2), we obtain the association rate constant k 1 = 1.5 ± 0.2 μM −1 s −1 and the dissociation rate constant k −1 = 6.9 ± 0.7 s −1 .

    Article Snippet: BODIPY-FL-ATP was purchased from Thermo Fisher Scientific (A12410, America).

    Techniques: Binding Assay, Fluorescence