exonuclease deficient dna polymerase  (New England Biolabs)

Journal: OncoTargets and therapy

Article Title: Ablation of MCM10 using CRISPR/Cas9 restrains the growth and migration of esophageal squamous cell carcinoma cells through inhibition of Akt signaling

doi: 10.2147/OTT.S157025

Figure Lengend Snippet: Deletion of MCM10 through CRISPR/Cas9 technology. Notes: ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

Article Snippet: After treatment with T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h, the resulting fragments were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

Techniques: CRISPR, Knock-Out, Construct, Transfection, Fluorescence, Sequencing, Clone Assay, Derivative Assay, Polymerase Chain Reaction, Amplification, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Migration, In Vitro, Wound Healing Assay

Journal: Sensors (Basel, Switzerland)

Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

doi: 10.3390/s19010077

Figure Lengend Snippet: Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), Klenow Fragment exo- (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.

Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

Techniques: Fluorescence

Journal: Sensors (Basel, Switzerland)

Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

doi: 10.3390/s19010077

Figure Lengend Snippet: The fluorescence spectrum of the developed sensing system is collected in a blank control sample (curve “a”); in the presence of aptamer DNA, an MB, polymerase, and nicking endonuclease, but without PDGF-BB (curve “b”); in the presence of PDGF-BB, aptamer DNA, and an MB (curve “c”); in the presence of PDGF-BB, aptamer DNA, an MB, and polymerase (curve “d”); and in the presence of PDGF-BB, aptamer DNA, MB, polymerase, and nicking endonuclease Nt.BbvCI (curve “e”). The reaction is in an NEB buffer (pH 7.9) at 37 °C for 2 h containing 1 μM aptamer DNA, 1 μM MB, 2.5 μM primer, 100 ng/mL PDGF-BB, 1 U/μL Klenow Fragment exo-, 2 mM dNTPs, and 1 U/μL Nt.BbvCI.

Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

doi: 10.1093/nar/gkn575

Figure Lengend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

Article Snippet: Primer extension with Klenow fragment of DNA polymerase Primer extension experiments using large fragment (Klenow) of DNA polymerase I (New England Biolabs) were performed at 25°C using the HIV-1 tat reverse primer (5′-AATACTATGGTCCACACAACTATTGCT-3′) that was unmodified or contained a single OXP modification.

Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

Journal: Nature

Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

doi: 10.1038/nature25449

Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

Techniques: Inhibition, Immunofluorescence, Staining