Exonuclease Deficient Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach"
Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach
Journal: PLoS ONE
Figure Legend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.
Techniques Used: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing
2) Product Images from "Single-stranded nucleic acid sensing and coacervation by linker histone H1"
Article Title: Single-stranded nucleic acid sensing and coacervation by linker histone H1
Figure Legend Snippet: H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.
Techniques Used: Fluorescence, Microscopy, Binding Assay, Imaging, Recombinase Polymerase Amplification
3) Product Images from "Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance"
Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance
Journal: Nucleic Acids Research
Figure Legend 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.
Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation
4) Product Images from "Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase"
Article Title: Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase
Figure Legend Snippet: CMG Undergoes Directional Translocation on ssDNA (A) Schematic of the experimental setup. Individual DNA tethers were formed in channels 1–3 separated by laminar flow containing streptavidin-coated beads, biotinylated λ-DNA, and buffer, respectively. They were subsequently moved to orthogonal channels 4 and 5 for protein loading and imaging. The illustration in the zoom-in box was not drawn to scale. (B) (Left) Cartoon and 2D scan of a tethered ssDNA loaded with multiple Cy3-CMGs. (Right) Representative kymographs of CMG movement in the presence of 1 mM ATP and 10 nM Mcm10 under 5 pN of tension. (C) Representative kymographs of Cy3-CMG (green) on ssDNA in the presence of Mcm10 but without ATP. (D) Distribution of CMG translocation rates on ssDNA in the absence (red) and presence (blue) of ATP ( n = 40 and 62, respectively). .
Techniques Used: Translocation Assay, Imaging
5) Product Images from "Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities"
Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities
Journal: Scientific Reports
Figure Legend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .
Techniques Used: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker