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Roche mgcl2
PCR optimization on mouse testis cDNA by <t>MgCl2</t> gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).
Mgcl2, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 2919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Producing Recombinant mTEX101; a Murine Testis Specific Protein"

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

Journal: Journal of Reproduction & Infertility

doi:

PCR optimization on mouse testis cDNA by MgCl2 gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).
Figure Legend Snippet: PCR optimization on mouse testis cDNA by MgCl2 gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).

Techniques Used: Polymerase Chain Reaction, Negative Control

2) Product Images from "Rapid and simple comparison of messenger RNA levels using real-time PCR"

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

Journal: Biological Procedures Online

doi: 10.1251/bpo114

MgCl2: Melt® results.
Figure Legend Snippet: MgCl2: Melt® results.

Techniques Used:

Optimization of MgCl2 concentration in samples.
Figure Legend Snippet: Optimization of MgCl2 concentration in samples.

Techniques Used: Concentration Assay

Related Articles

Clone Assay:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. The EMAP-II cDNA product was then cloned into the pCR2.1-TOPO vector (Invitrogen) and run-off transcripts in the sense and anti-sense direction labeled in vitro (Promega) with biotin (Sigma).

Amplification:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. Amplification proceeded for 40 cycles at 94°C for 1 minute, 60°C for 1 minute, 72°C for 2 minutes, and an additional extension period of 5 minutes at 72°C.

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: .. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA. ..

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
Article Snippet: Each sample consisted of: 50 ng cDNA, 3.0 mM MgCl2 , 200 μM dNTP (Roche, Nonnenwald, Germany), 500 nM of primers, 2 μl 10X PCR buffer (10X PCR buffer is: 100 mM Tris-Hcl pH 8.5, 500 mM KCl, 1.5% Triton X-100), and 0.1 unit of Taq polymerase (Amersham Biosciences), in a total reaction volume of 20 μl ( ). .. Amplification conditions included an initial step at 95°C (3 min), followed by 25 repetitions of the following cycle: 95°C (30 sec) gradient temperatures between 56° to 62°C (30 sec), 72°C (20 sec), then concluded by a step at 72°C for 5 min. Amplicons were migrated and visualized on 2% agarose gels in order to confirm optimal melting temperature as determined by the brightest band.

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: .. The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA. .. The quantification of the unknown samples was performed by LightCycler Relative Quantification Software, version 3.3 (Roche Diagnostics, Basel, Switzerland).

Synthesized:

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: Primers were designed by using Primer Designer (Scientific and Education Software) and were synthesized by the University of Ottawa Biotechnology Research Institute. .. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA.

Quantitative RT-PCR:

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: Paragraph title: 4.11. Quantitative RT-PCR (qRT-PCR) ... The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA.

SYBR Green Assay:

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: .. The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA. .. The quantification of the unknown samples was performed by LightCycler Relative Quantification Software, version 3.3 (Roche Diagnostics, Basel, Switzerland).

Random Hexamer Labeling:

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: One microgram of the total RNA was reversetranscribed to cDNA by using 200U of Molony Murine Leukemia Virus (RTM-MULV) reverse transcriptase enzyme (Fermentas, Vilnius, Lithuania), and 20pmol of random hexamer primers (Cybergene, Stockholm, Sweden). .. PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

Expressing:

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: Paragraph title: High throughput expression and purification of proteins ... Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol.

BIA-KA:

Article Title: Transgenic Rescue of ataxia Mice with Neuronal-Specific Expression of Ubiquitin-Specific Protease 14
Article Snippet: Tissues were homogenized in 1–3 ml of homogenization buffer containing the following (in m m ): 50 Tris, pH 7.5, 150 NaCl, 5 MgCl2 , 2 N -ethylmaleimide, plus 0.5% SDS and Complete protease inhibitors from Roche (Indianapolis, IN). .. Protein concentrations were determined by using the bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, IL).

Western Blot:

Article Title: Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium
Article Snippet: Paragraph title: Western blot and Phosphorylation assays ... Then the cells were stimulated with 10ng/ml IL-1β with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 10 min, or 2ng/ml TNFα with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 15 min. After treatment, the cells were washed with ice-cold PBS and lysed in 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 10% Glycerol, 1% NP-40, with protease and phosphatase inhibitors (Roche).

Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Complement Factor D protects mice from ethanol-induced inflammation and liver injury
Article Snippet: Paragraph title: Liver Homogenates and Western Blotting ... Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN).

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: .. Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). ..

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
Article Snippet: Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. To examine the interaction of Prdx1 with ASK1, p38 MAPK, and c-JNK, immune complexes were analyzed on Western blots.

Sequencing:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. Anti-sense sequence: 5′-TCATTTGATTCCACTGTTGCTCATGG-3′; sense sequence: 5′-AGGATGGACTCTAAGCCAATAGAT-3′.

Inverse PCR:

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: Oligonucleotide primers used for PCR and inverse PCR (IPCR) in this study are listed in Table . .. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA.

Immunoprecipitation:

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

Protease Inhibitor:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: .. HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The reaction products were phenol/chloroform extracted and ethanol precipitated using pellet paint (Novagen) plus tRNA.

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: .. Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol. .. The lysates were then transferred to Whatman 96-well glass fiber-filter plates (Millipore Sigma) and filtered by spinning.

Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Complement Factor D protects mice from ethanol-induced inflammation and liver injury
Article Snippet: .. Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). .. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Hercules, CA), and samples were denatured at 95°C in Laemmli buffer.

Article Title: A CRY–BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis
Article Snippet: .. Approximately 4 g seedlings were ground in liquid nitrogen and homogenized in 40 mL nuclei isolation and X-linking buffer [0.4 m sucrose, 10 mM HEPEs (pH 8.0), 5 mM KCl, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM PMSF, 1× Protease inhibitor cocktail (Roche) and 1% formaldehyde] for 20 min at room temperature. ..

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: .. Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). ..

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

In Situ Hybridization:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: Paragraph title: In Situ Hybridization ... The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche).

Generated:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: A 540-bp cDNA product corresponding to the transcript encoding EMAP-II was generated by polymerase chain reaction. .. The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche).

Article Title: Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium
Article Snippet: Then the cells were stimulated with 10ng/ml IL-1β with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 10 min, or 2ng/ml TNFα with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 15 min. After treatment, the cells were washed with ice-cold PBS and lysed in 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 10% Glycerol, 1% NP-40, with protease and phosphatase inhibitors (Roche). .. For RhoA, ARF6, Rac1 /cdc42 and R-Ras activation assays, crude total cell lysate were generated and GTP-RhoA, ARF6, Rac1/ cdc42 and R-Ras were precipitated with Rhotekin-RBD (Millipore), GGA3-PBD (Cell Biolabs), PAK-1-PBD (Millipore) and Raf-1 RBD respectively.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: Paragraph title: RT PCR ... PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

Aminoacylation Assay:

Article Title: A human leucyl-tRNA synthetase as an anticancer target
Article Snippet: Paragraph title: Aminoacylation assay ... Briefly, the experiments were performed in 70 μL reaction mixtures containing 50 mM HEPES–KOH (pH 7.8), 5 mM MgCl2 , 45 mM KCl, 1 mM DTT, 0.02% BSA (W/V), 0.4 mg/mL of brewer’s yeast tRNA (Roche, Basel, Switzerland), 10 μM Leu (PerkinElmer, Waltham, MA, USA), and 2 nM tb LARS (or hs LARS).

Recombinant:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: .. HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The reaction products were phenol/chloroform extracted and ethanol precipitated using pellet paint (Novagen) plus tRNA.

DC Protein Assay:

Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Complement Factor D protects mice from ethanol-induced inflammation and liver injury
Article Snippet: Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). .. Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN).

Nucleic Acid Electrophoresis:

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). .. Proteins were separated in 4–12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Life Technologies) and then transferred to PVDF membranes (GE Healthcare) for immunodetection.

Radioactivity:

Article Title: A human leucyl-tRNA synthetase as an anticancer target
Article Snippet: Briefly, the experiments were performed in 70 μL reaction mixtures containing 50 mM HEPES–KOH (pH 7.8), 5 mM MgCl2 , 45 mM KCl, 1 mM DTT, 0.02% BSA (W/V), 0.4 mg/mL of brewer’s yeast tRNA (Roche, Basel, Switzerland), 10 μM Leu (PerkinElmer, Waltham, MA, USA), and 2 nM tb LARS (or hs LARS). .. The filter was then dried under an infrared heat lamp, and the radioactivity of the precipitate was quantified using a scintillation counter (Beckman Coulter, Irvine, CA, USA).

Isolation:

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: RT PCR Total RNA was isolated from male gonadal organs (four to 6 week-old mice from Pasteur Institute of Iran). .. PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

Article Title: A CRY–BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis
Article Snippet: .. Approximately 4 g seedlings were ground in liquid nitrogen and homogenized in 40 mL nuclei isolation and X-linking buffer [0.4 m sucrose, 10 mM HEPEs (pH 8.0), 5 mM KCl, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM PMSF, 1× Protease inhibitor cocktail (Roche) and 1% formaldehyde] for 20 min at room temperature. ..

Article Title: Transgenic Rescue of ataxia Mice with Neuronal-Specific Expression of Ubiquitin-Specific Protease 14
Article Snippet: Paragraph title: Isolation of mouse proteins. ... Tissues were homogenized in 1–3 ml of homogenization buffer containing the following (in m m ): 50 Tris, pH 7.5, 150 NaCl, 5 MgCl2 , 2 N -ethylmaleimide, plus 0.5% SDS and Complete protease inhibitors from Roche (Indianapolis, IN).

Size-exclusion Chromatography:

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
Article Snippet: Each sample consisted of: 50 ng cDNA, 3.0 mM MgCl2 , 200 μM dNTP (Roche, Nonnenwald, Germany), 500 nM of primers, 2 μl 10X PCR buffer (10X PCR buffer is: 100 mM Tris-Hcl pH 8.5, 500 mM KCl, 1.5% Triton X-100), and 0.1 unit of Taq polymerase (Amersham Biosciences), in a total reaction volume of 20 μl ( ). .. Amplification conditions included an initial step at 95°C (3 min), followed by 25 repetitions of the following cycle: 95°C (30 sec) gradient temperatures between 56° to 62°C (30 sec), 72°C (20 sec), then concluded by a step at 72°C for 5 min. Amplicons were migrated and visualized on 2% agarose gels in order to confirm optimal melting temperature as determined by the brightest band.

Labeling:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The purified DNA was then digested with the Sfc I restriction endonuclease (NEB) for 2 h at 25°C followed by labeling with [α-32 P]-TTP for 2 h at 25°C using Klenow (NEB).

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. The EMAP-II cDNA product was then cloned into the pCR2.1-TOPO vector (Invitrogen) and run-off transcripts in the sense and anti-sense direction labeled in vitro (Promega) with biotin (Sigma).

Mouse Assay:

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: RT PCR Total RNA was isolated from male gonadal organs (four to 6 week-old mice from Pasteur Institute of Iran). .. PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

Article Title: Transgenic Rescue of ataxia Mice with Neuronal-Specific Expression of Ubiquitin-Specific Protease 14
Article Snippet: Mice 4–6 weeks of age and of appropriate genotype were killed by CO2 asphyxiation. .. Tissues were homogenized in 1–3 ml of homogenization buffer containing the following (in m m ): 50 Tris, pH 7.5, 150 NaCl, 5 MgCl2 , 2 N -ethylmaleimide, plus 0.5% SDS and Complete protease inhibitors from Roche (Indianapolis, IN).

Polymerase Chain Reaction:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: .. The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. Amplification proceeded for 40 cycles at 94°C for 1 minute, 60°C for 1 minute, 72°C for 2 minutes, and an additional extension period of 5 minutes at 72°C.

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: .. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA. ..

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: .. PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany). .. PCR oligonucleotide primers were designed based on the mTEX101 ORF.

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
Article Snippet: .. Each sample consisted of: 50 ng cDNA, 3.0 mM MgCl2 , 200 μM dNTP (Roche, Nonnenwald, Germany), 500 nM of primers, 2 μl 10X PCR buffer (10X PCR buffer is: 100 mM Tris-Hcl pH 8.5, 500 mM KCl, 1.5% Triton X-100), and 0.1 unit of Taq polymerase (Amersham Biosciences), in a total reaction volume of 20 μl ( ). ..

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA. .. PCR reactions were prepared in duplicate and heated to 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 15 s, and extension at 72 °C for 20 s. Standard curves (cycle threshold values versus template concentration) were prepared for each target gene and for the endogenous reference (GAPDH) in each sample.

Immunodetection:

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). .. Proteins were separated in 4–12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Life Technologies) and then transferred to PVDF membranes (GE Healthcare) for immunodetection.

Concentration Assay:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: .. HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The reaction products were phenol/chloroform extracted and ethanol precipitated using pellet paint (Novagen) plus tRNA.

Article Title: Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans
Article Snippet: .. After cooling to room temperature, iodoacetamide was added to a final concentration of 7 mM, and the sample was vortexed and incubated at room temperature in the dark for 30 min. After DTT was added to a final concentration of 7 mM, 10 μg LysC was added, and the samples were vortexed and incubated at 37 °C for 4 h. The urea concentration was subsequently diluted to 1.5 M by the addition of 40 mM Tris (pH 8.0), 1 mM CaCl2 , and 2 mM MgCl2. .. After acidifying with 10% trifluoroacetic acid (TFA) (final concentration, ~1%), the peptides were desalted by solid phase extraction using 50 mg tC18 Sep-Pak cartridges (Waters) and dried in a speed vacuum.

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
Article Snippet: RNA concentration was measured by a BioPhotometer (Eppendorf, Hamburg, Germany) at 260nm . .. PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: IPTG (Thermo Fisher, Waltham, MA) was then added to a final concentration of 1 mM and the culture was incubated for another 2 hours for most proteins, or 1.5 hours for predicted membrane and secretory proteins. .. Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol.

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA. .. PCR reactions were prepared in duplicate and heated to 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 15 s, and extension at 72 °C for 20 s. Standard curves (cycle threshold values versus template concentration) were prepared for each target gene and for the endogenous reference (GAPDH) in each sample.

Purification:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The purified DNA was then digested with the Sfc I restriction endonuclease (NEB) for 2 h at 25°C followed by labeling with [α-32 P]-TTP for 2 h at 25°C using Klenow (NEB).

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: Paragraph title: High throughput expression and purification of proteins ... Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol.

Chromatin Immunoprecipitation:

Article Title: A CRY–BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) assays ... Approximately 4 g seedlings were ground in liquid nitrogen and homogenized in 40 mL nuclei isolation and X-linking buffer [0.4 m sucrose, 10 mM HEPEs (pH 8.0), 5 mM KCl, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM PMSF, 1× Protease inhibitor cocktail (Roche) and 1% formaldehyde] for 20 min at room temperature.

SDS Page:

Article Title: Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium
Article Snippet: Then the cells were stimulated with 10ng/ml IL-1β with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 10 min, or 2ng/ml TNFα with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 15 min. After treatment, the cells were washed with ice-cold PBS and lysed in 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 10% Glycerol, 1% NP-40, with protease and phosphatase inhibitors (Roche). .. Lysates were combined with 2X sample buffer, separated by SDS-PAGE and probed with antibodies to phospho-VE-cadehrin Y731 (Invitrogen), phospho-Src Y418 (Cell signaling), VE- cadherin (Cell signaling), Src (Cell signaling), FoxO1 (Cell signaling) and CYP24 (Thermo) at 1:1000.

Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Complement Factor D protects mice from ethanol-induced inflammation and liver injury
Article Snippet: Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). .. Samples were separated on SDS-PAGE gels, transferred to polyvinylidene fluoride membranes with a semidry transfer apparatus (Bio-Rad), and blocked in 3% bovine serum albumin.

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). .. Proteins were separated in 4–12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (Life Technologies) and then transferred to PVDF membranes (GE Healthcare) for immunodetection.

Plasmid Preparation:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. The EMAP-II cDNA product was then cloned into the pCR2.1-TOPO vector (Invitrogen) and run-off transcripts in the sense and anti-sense direction labeled in vitro (Promega) with biotin (Sigma).

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA. .. The concentrations of plasmid DNA templates were adjusted to 0.01 μg/ml with ddH2 O for IPCRs.

Software:

Article Title: Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors
Article Snippet: Primers were designed by using Primer Designer (Scientific and Education Software) and were synthesized by the University of Ottawa Biotechnology Research Institute. .. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H2 O (ddH2 O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl2 (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA.

Article Title: Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization
Article Snippet: The amplification was carried out in a total volume of 20 μL, containing 0.5 mM of each primer, 4 mM MgCl2 , 20 μL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) and 20 μL of 1:10 diluted cDNA. .. The quantification of the unknown samples was performed by LightCycler Relative Quantification Software, version 3.3 (Roche Diagnostics, Basel, Switzerland).

Binding Assay:

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol. .. The plates were then sealed at the top and rotated for 1 hour at 4°C to allow binding of fusion proteins to the beads.

In Vitro:

Article Title: Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo
Article Snippet: The polymerase chain reaction mixture contained 0.5 nmol/L dATP, 0.5 nmol/L dCTP, 0.5 nmol/L dGTP, 0.5 nmol/L dTTP, 100 pmol/L each forward and reverse primer, 10 mmol/L Tris-HCL, pH 8.3, 1.5 mmol/L MgCl2 , 50 mmol/L KCl, and 1 U Taq polymerase (Roche). .. The EMAP-II cDNA product was then cloned into the pCR2.1-TOPO vector (Invitrogen) and run-off transcripts in the sense and anti-sense direction labeled in vitro (Promega) with biotin (Sigma).

Homogenization:

Article Title: Transgenic Rescue of ataxia Mice with Neuronal-Specific Expression of Ubiquitin-Specific Protease 14
Article Snippet: .. Tissues were homogenized in 1–3 ml of homogenization buffer containing the following (in m m ): 50 Tris, pH 7.5, 150 NaCl, 5 MgCl2 , 2 N -ethylmaleimide, plus 0.5% SDS and Complete protease inhibitors from Roche (Indianapolis, IN). .. Protein concentrations were determined by using the bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, IL).

Incubation:

Article Title: Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?
Article Snippet: .. HBV core DNA (600 pg RC DNA equivalent) extracted from cytoplasmic nucleocapsids with proteinase K digestion was incubated with recombinant Tdp2 (final concentration, 320 nM) at 37°C for 1 hr, in 1x Tdp2 buffer (25 mM Tris-HCl, pH 8.0, 130 mM KCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2 ) containing 1X Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (P.I.s, Roche). .. The reaction products were phenol/chloroform extracted and ethanol precipitated using pellet paint (Novagen) plus tRNA.

Article Title: Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans
Article Snippet: .. After cooling to room temperature, iodoacetamide was added to a final concentration of 7 mM, and the sample was vortexed and incubated at room temperature in the dark for 30 min. After DTT was added to a final concentration of 7 mM, 10 μg LysC was added, and the samples were vortexed and incubated at 37 °C for 4 h. The urea concentration was subsequently diluted to 1.5 M by the addition of 40 mM Tris (pH 8.0), 1 mM CaCl2 , and 2 mM MgCl2. .. After acidifying with 10% trifluoroacetic acid (TFA) (final concentration, ~1%), the peptides were desalted by solid phase extraction using 50 mg tC18 Sep-Pak cartridges (Waters) and dried in a speed vacuum.

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: IPTG (Thermo Fisher, Waltham, MA) was then added to a final concentration of 1 mM and the culture was incubated for another 2 hours for most proteins, or 1.5 hours for predicted membrane and secretory proteins. .. Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol.

Article Title: A human leucyl-tRNA synthetase as an anticancer target
Article Snippet: Briefly, the experiments were performed in 70 μL reaction mixtures containing 50 mM HEPES–KOH (pH 7.8), 5 mM MgCl2 , 45 mM KCl, 1 mM DTT, 0.02% BSA (W/V), 0.4 mg/mL of brewer’s yeast tRNA (Roche, Basel, Switzerland), 10 μM Leu (PerkinElmer, Waltham, MA, USA), and 2 nM tb LARS (or hs LARS). .. The reaction mixture was incubated at 37°C for 20 minutes and then initiated by adding 4 mM adenosine triphosphate.

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

Activation Assay:

Article Title: Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium
Article Snippet: Then the cells were stimulated with 10ng/ml IL-1β with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 10 min, or 2ng/ml TNFα with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 15 min. After treatment, the cells were washed with ice-cold PBS and lysed in 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 10% Glycerol, 1% NP-40, with protease and phosphatase inhibitors (Roche). .. For RhoA, ARF6, Rac1 /cdc42 and R-Ras activation assays, crude total cell lysate were generated and GTP-RhoA, ARF6, Rac1/ cdc42 and R-Ras were precipitated with Rhotekin-RBD (Millipore), GGA3-PBD (Cell Biolabs), PAK-1-PBD (Millipore) and Raf-1 RBD respectively.

Fractionation:

Article Title: Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA
Article Snippet: .. Western blot analyses For fractionation, cells were treated with Cytoskeleton (CSK) buffer (0.5% Triton X-100; 10 mM, 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.8; 100 mM NaCl; 300 mM sucrose; 3 mM MgCl2; 1 mM EGTA; 1mM PMSF) plus protease inhibitor cocktail (Roche). ..

High Throughput Screening Assay:

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: Paragraph title: High throughput expression and purification of proteins ... Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol.

Lysis:

Article Title: Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium
Article Snippet: Then the cells were stimulated with 10ng/ml IL-1β with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 10 min, or 2ng/ml TNFα with 10μm D3 (Tocris), 7-DHC (Sigma) or vehicle (0.5% DMSO) for 15 min. After treatment, the cells were washed with ice-cold PBS and lysed in 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 10% Glycerol, 1% NP-40, with protease and phosphatase inhibitors (Roche). .. Following three washes with lysis buffer, bound proteins were eluted with 2X sample buffer.

Article Title: A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens
Article Snippet: .. Cell pellets in the 24-well plates were resuspended and lysed in 160 μL of Novagen BugBuster lysis buffer (Millipore Sigma, Burlington, MA) supplemented with 100 μg/mL lysozyme, 8 μg/mL, DNase 2 mM MgCl2 , 10 mM NaF, 1 mM Na3 VO4 , protease inhibitor tablets (Roche Applied Science, Indianapolis, IN), 500 mM PMSF, 0.1% β-mercaptoethanol, and 10% glycerol. .. The lysates were then transferred to Whatman 96-well glass fiber-filter plates (Millipore Sigma) and filtered by spinning.

Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Complement Factor D protects mice from ethanol-induced inflammation and liver injury
Article Snippet: .. Protein lysates were made from frozen liver in lysis buffer containing: 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM MgCl2 , 2 mM EGTA, 10 mM NaF, 1 mM PMSF, 1 mM Na3 (VO3 )4 , 12.5 mM β-glycerol phosphate, 2 mM DTT, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). .. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Hercules, CA), and samples were denatured at 95°C in Laemmli buffer.

Article Title: A CRY–BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis
Article Snippet: Approximately 4 g seedlings were ground in liquid nitrogen and homogenized in 40 mL nuclei isolation and X-linking buffer [0.4 m sucrose, 10 mM HEPEs (pH 8.0), 5 mM KCl, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM PMSF, 1× Protease inhibitor cocktail (Roche) and 1% formaldehyde] for 20 min at room temperature. .. The pellet was suspended in 400 μl of nuclei lysis buffer [50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF and 1× Protease inhibitor cocktail], and the nuclei was lysed by pipetting up and down slowly at room temperature.

Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

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  • mgcl2  (Roche)
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    Roche mgcl2
    PCR optimization on mouse testis cDNA by <t>MgCl2</t> gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).
    Mgcl2, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 2919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mgcl2/product/Roche
    Average 98 stars, based on 2919 article reviews
    Price from $9.99 to $1999.99
    mgcl2 - by Bioz Stars, 2020-02
    98/100 stars
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    94
    Roche m mgcl2
    PCR optimization on mouse testis cDNA by <t>MgCl2</t> gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).
    M Mgcl2, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mgcl2/product/Roche
    Average 94 stars, based on 282 article reviews
    Price from $9.99 to $1999.99
    m mgcl2 - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    Image Search Results


    PCR optimization on mouse testis cDNA by MgCl2 gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).

    Journal: Journal of Reproduction & Infertility

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

    doi:

    Figure Lengend Snippet: PCR optimization on mouse testis cDNA by MgCl2 gradients. 1-4: MgCl2 concentrations from 1 to 4 mM, respectively, 5: 1 Kb DNA ladder, 6: negative control (no DNA).

    Article Snippet: PCR amplifications for mTEX101 transcript was performed in a volume of 25µl using 10-20ng of testis cDNA, forward and reverse primers (10pmol each), specific for mTEX101 transcript, 10X PCR buffer (2.5µl ), dNTP mixture (0.2mM each), 1mM MgCl2, and 1 unit of Taq DNA polymerase (Roche, Mannheim, Germany).

    Techniques: Polymerase Chain Reaction, Negative Control

    MgCl2: Melt® results.

    Journal: Biological Procedures Online

    Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

    doi: 10.1251/bpo114

    Figure Lengend Snippet: MgCl2: Melt® results.

    Article Snippet: Each sample consisted of: 50 ng cDNA, 3.0 mM MgCl2 , 200 μM dNTP (Roche, Nonnenwald, Germany), 500 nM of primers, 2 μl 10X PCR buffer (10X PCR buffer is: 100 mM Tris-Hcl pH 8.5, 500 mM KCl, 1.5% Triton X-100), and 0.1 unit of Taq polymerase (Amersham Biosciences), in a total reaction volume of 20 μl ( ).

    Techniques:

    Optimization of MgCl2 concentration in samples.

    Journal: Biological Procedures Online

    Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

    doi: 10.1251/bpo114

    Figure Lengend Snippet: Optimization of MgCl2 concentration in samples.

    Article Snippet: Each sample consisted of: 50 ng cDNA, 3.0 mM MgCl2 , 200 μM dNTP (Roche, Nonnenwald, Germany), 500 nM of primers, 2 μl 10X PCR buffer (10X PCR buffer is: 100 mM Tris-Hcl pH 8.5, 500 mM KCl, 1.5% Triton X-100), and 0.1 unit of Taq polymerase (Amersham Biosciences), in a total reaction volume of 20 μl ( ).

    Techniques: Concentration Assay

    ATPase activity of digitonin solubilized E. coli F 1 F o ATP synthase. E. coli F 1 F o ATP synthase purified in digitonin was incubated at room temperature and assessed for ATPase activity at 37°C was measured at 0, 1, 2, 3, 4 and 8 hr time points. ATP regeneration assays were performed as described in. ATPase activity normalized to 80 s −1 showed little change over 8 hr. The rate of ATP hydrolysis was measured with an ATP-regenerating coupled assay that resulted in a final concentration of 50 mM Tris–HCl (pH 8.0), 10 mM KCl, 2.5 mM phosphoenolpyruvate, 0.3 mM NADH, 50 μg/ml pyruvate kinase, 50 μg/ml lactate dehydrogenase, and 2 mM MgCl2 and 1 mM ATP. The rate was determined from the linear change in absorbance at 340 nm, once activity achieved steady-state.

    Journal: eLife

    Article Title: Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

    doi: 10.7554/eLife.43864

    Figure Lengend Snippet: ATPase activity of digitonin solubilized E. coli F 1 F o ATP synthase. E. coli F 1 F o ATP synthase purified in digitonin was incubated at room temperature and assessed for ATPase activity at 37°C was measured at 0, 1, 2, 3, 4 and 8 hr time points. ATP regeneration assays were performed as described in. ATPase activity normalized to 80 s −1 showed little change over 8 hr. The rate of ATP hydrolysis was measured with an ATP-regenerating coupled assay that resulted in a final concentration of 50 mM Tris–HCl (pH 8.0), 10 mM KCl, 2.5 mM phosphoenolpyruvate, 0.3 mM NADH, 50 μg/ml pyruvate kinase, 50 μg/ml lactate dehydrogenase, and 2 mM MgCl2 and 1 mM ATP. The rate was determined from the linear change in absorbance at 340 nm, once activity achieved steady-state.

    Article Snippet: The ATP synthase complex was extracted from membranes at 4°C for 1 hr by resuspending the pellet in extraction buffer consisting of 20 mM Tris/Cl, pH 8.0, 300 mM NaCl, 2 mM MgCl2 , 100 mM sucrose, 20 mM imidazole, 10% glycerol, 4 mM digitonin and EDTA-free protease inhibitor tablets (Roche) [~1.5 hr for resuspension].

    Techniques: Activity Assay, Purification, Incubation, Concentration Assay