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    NEB Express Competent E coli High Efficiency
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    NEB Express Competent E coli High Efficiency 20x0 05 ml
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    c2523h
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    Competent Bacteria
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    1 ml
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    New England Biolabs e coli ssb
    NEB Express Competent E coli High Efficiency
    NEB Express Competent E coli High Efficiency 20x0 05 ml
    https://www.bioz.com/result/e coli ssb/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli ssb - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2"

    Article Title: RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15609-x

    Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.
    Figure Legend Snippet: Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.

    Techniques Used: Incubation, Purification, Electron Microscopy

    2) Product Images from "Semliki Forest virus strongly reduces mosquito host defence signaling"

    Article Title: Semliki Forest virus strongly reduces mosquito host defence signaling

    Journal: Insect Molecular Biology

    doi: 10.1111/j.1365-2583.2008.00834.x

    Activation of host cell defence signaling inhibits SFV4 RNA replication. U4.4 cells were treated with heat-inactivated Escherichia coli for 1 h (to stimulate signaling pathways involving STAT/IMD) or mock-treated, then infected with SFV4 (m.o.i. 1) for 12 h before RNA isolation. Viral genome copy numbers, which also act as viral polyprotein mRNA and are thus linked to viral gene expression, were measured by real time quantitative PCR targeting a region of nsP3. RNAs from three independent biological replicates (for both non-stimulated and stimulated cells) were reverse transcribed in triplicate; each reverse transcription reaction was then amplified in triplicate. Each bar represents the average of all measurements from a representative real time quantitative PCR experiment (see Experiemental procedures); error bars represent the standard deviation. Every experiment was repeated at least twice under the same conditions.
    Figure Legend Snippet: Activation of host cell defence signaling inhibits SFV4 RNA replication. U4.4 cells were treated with heat-inactivated Escherichia coli for 1 h (to stimulate signaling pathways involving STAT/IMD) or mock-treated, then infected with SFV4 (m.o.i. 1) for 12 h before RNA isolation. Viral genome copy numbers, which also act as viral polyprotein mRNA and are thus linked to viral gene expression, were measured by real time quantitative PCR targeting a region of nsP3. RNAs from three independent biological replicates (for both non-stimulated and stimulated cells) were reverse transcribed in triplicate; each reverse transcription reaction was then amplified in triplicate. Each bar represents the average of all measurements from a representative real time quantitative PCR experiment (see Experiemental procedures); error bars represent the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Techniques Used: Activation Assay, Radial Immuno Diffusion, Infection, Isolation, Activated Clotting Time Assay, Expressing, Real-time Polymerase Chain Reaction, Amplification, Standard Deviation

    Effects of host cell defence signaling on SFV gene expression. (A) Recombinant SFV4-derived viruses expressing Renilla luciferase ( Rluc ) from the non-structural (SFV4(3H)- Rluc ) or structural region of the genome (SFV4-st Rluc ). (B) U4.4 cells were stimulated with heat-inactivated Escherichia coli for 1 h or mock-treated, then infected (m.o.i. 1) with recombinant SFVs. Luciferase expression was measured at 12 h p.i. (C) U4.4 cells were transfected with constitutively active Toll ΔLRR receptor (pJL195) or mock-stimulated (transfection of pIB-V5/His; empty insect cell expression vector) for 24 h then infected with (m.o.i. 1) SFV4(3H)- Rluc or SFV4-st Rluc . Cells were lysed 12 h p.i. and luciferase activities determined. Each bar represents the mean of three independent biological replicates; error bars indicate the standard deviation. Every experiment was repeated at least twice under the same conditions.
    Figure Legend Snippet: Effects of host cell defence signaling on SFV gene expression. (A) Recombinant SFV4-derived viruses expressing Renilla luciferase ( Rluc ) from the non-structural (SFV4(3H)- Rluc ) or structural region of the genome (SFV4-st Rluc ). (B) U4.4 cells were stimulated with heat-inactivated Escherichia coli for 1 h or mock-treated, then infected (m.o.i. 1) with recombinant SFVs. Luciferase expression was measured at 12 h p.i. (C) U4.4 cells were transfected with constitutively active Toll ΔLRR receptor (pJL195) or mock-stimulated (transfection of pIB-V5/His; empty insect cell expression vector) for 24 h then infected with (m.o.i. 1) SFV4(3H)- Rluc or SFV4-st Rluc . Cells were lysed 12 h p.i. and luciferase activities determined. Each bar represents the mean of three independent biological replicates; error bars indicate the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Techniques Used: Expressing, Recombinant, Derivative Assay, Luciferase, Infection, Transfection, Plasmid Preparation, Standard Deviation

    3) Product Images from "Transcription factor YcjW controls the emergency H2S production in E. coli"

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10785-x

    E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p
    Figure Legend Snippet: E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Techniques Used: Time-Kill Assay, Staining

    4) Product Images from "Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis"

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1719497115

    ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.
    Figure Legend Snippet: ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Techniques Used: Generated, Incubation, Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Fluorescence

    FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.
    Figure Legend Snippet: FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Techniques Used: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence

    Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.
    Figure Legend Snippet: Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.

    Techniques Used: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: When several bands were observed, PCR products were purified using the High Pure Purification kit (Roche, Mannhein, Germany). .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Amplification:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Clone Assay:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: When several bands were observed, PCR products were purified using the High Pure Purification kit (Roche, Mannhein, Germany). .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Plasmid Preparation:

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli
    Article Snippet: .. To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB). .. Plasmid pLLSN3 was generated as above, except ycjW was PCR amplified from mstA-sup.

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: When several bands were observed, PCR products were purified using the High Pure Purification kit (Roche, Mannhein, Germany). .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA
    Article Snippet: .. Protein overexpression was carried out in the NEB Express E. coli strain (NEB) containing the relevant T7 expression plasmid ( ). .. Expression from the T7 promoter was induced at mid-exponential growth phase with 0.2 mM IPTG at 20°C overnight.

    other:

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis
    Article Snippet: Validation of DNA Substrates Containing N7-meG and Me-FAPy-G. DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Transformation Assay:

    Article Title: Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2
    Article Snippet: When several bands were observed, PCR products were purified using the High Pure Purification kit (Roche, Mannhein, Germany). .. Eluted DNA was inserted into the blunt pJET vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) and transformed into DH10β chemically competent Escherichia coli cells (NEB). .. Forward and reverse sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) using the same primers used for amplification or vector primers.

    Expressing:

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: .. Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease.

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA
    Article Snippet: .. Protein overexpression was carried out in the NEB Express E. coli strain (NEB) containing the relevant T7 expression plasmid ( ). .. Expression from the T7 promoter was induced at mid-exponential growth phase with 0.2 mM IPTG at 20°C overnight.

    Binding Assay:

    Article Title: Catch Bond Interaction between Cell-Surface Sulfatase Sulf1 and Glycosaminoglycans
    Article Snippet: .. For expression in Escherichia coli ( E. coli ), a pMAL-c5X plasmid (New England Biolabs, Ipswich, MA) encoding the maltose binding protein-HD (MBP-HD) fusion protein was used. .. MBP-HD consists of an N-terminal (MBP) sequence followed by the sequence of the HD from human Sulf1 (K417 -K735 ), as schematically depicted in a .

    Purification:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: .. Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease.

    Produced:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: .. Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease.

    Recombinant:

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons
    Article Snippet: .. Protein expression and purification Full antigens and their truncated form ( and ) were produced by recombinant expression in E. coli NEB Express strain (New England Biolabs, Ipswich, MA, USA). .. The expression vector was derived from the LacO-pQLinkN-construct containing a N-terminal Histidine-tag and a bdSUMO-domain fused to the protein of interest to increase protein solubility and allow cleavage with bdSUMO protease.

    Incubation:

    Article Title: RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2
    Article Snippet: DNA was purified on PCR clean-up columns (Macherey-Nagel) with NTB buffer. .. DNA was incubated with E. coli SSB (NEB) at a ratio of 6 μg SSB per μg DNA on ice for 10 min . .. Glutaraldehyde (Electron Microscopy Sciences) was added to a final concentration of 0.6% and samples incubated 10 min on ice.

    Article Title: Semliki Forest virus strongly reduces mosquito host defence signaling
    Article Snippet: Luciferase activities were measured using a Dual Luciferase assay kit (Promega) on a GloMax 20/20 Luminometer. .. Pathway stimulation with Escherichia coli To prepare working stocks of E. coli strain JM109 (New England Biolabs, Hitchim, UK), 1 µl of bacterial stock solution was added to 5 ml LB medium (without antibiotics), incubated at 37 °C/18 h, and centrifuged (4 °C, 2500 rpm/10 min). .. Cells were resuspended in 0.5 ml PBS, and bacteria inactivated by heating the suspension at 80 °C for 10 min. Fresh stocks were prepared for each experiment (concentration approx.

    Over Expression:

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA
    Article Snippet: .. Protein overexpression was carried out in the NEB Express E. coli strain (NEB) containing the relevant T7 expression plasmid ( ). .. Expression from the T7 promoter was induced at mid-exponential growth phase with 0.2 mM IPTG at 20°C overnight.

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    New England Biolabs e coli ssb
    Nuclease contamination cannot explain <t>RNA–DNA</t> hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. <t>SSB</t> protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.
    E Coli Ssb, 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
    https://www.bioz.com/result/e coli ssb/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli ssb - by Bioz Stars, 2021-03
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    Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.

    Journal: Nature Communications

    Article Title: RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

    doi: 10.1038/s41467-020-15609-x

    Figure Lengend Snippet: Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.

    Article Snippet: DNA was incubated with E. coli SSB (NEB) at a ratio of 6 μg SSB per μg DNA on ice for 10 min .

    Techniques: Incubation, Purification, Electron Microscopy

    Activation of host cell defence signaling inhibits SFV4 RNA replication. U4.4 cells were treated with heat-inactivated Escherichia coli for 1 h (to stimulate signaling pathways involving STAT/IMD) or mock-treated, then infected with SFV4 (m.o.i. 1) for 12 h before RNA isolation. Viral genome copy numbers, which also act as viral polyprotein mRNA and are thus linked to viral gene expression, were measured by real time quantitative PCR targeting a region of nsP3. RNAs from three independent biological replicates (for both non-stimulated and stimulated cells) were reverse transcribed in triplicate; each reverse transcription reaction was then amplified in triplicate. Each bar represents the average of all measurements from a representative real time quantitative PCR experiment (see Experiemental procedures); error bars represent the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Journal: Insect Molecular Biology

    Article Title: Semliki Forest virus strongly reduces mosquito host defence signaling

    doi: 10.1111/j.1365-2583.2008.00834.x

    Figure Lengend Snippet: Activation of host cell defence signaling inhibits SFV4 RNA replication. U4.4 cells were treated with heat-inactivated Escherichia coli for 1 h (to stimulate signaling pathways involving STAT/IMD) or mock-treated, then infected with SFV4 (m.o.i. 1) for 12 h before RNA isolation. Viral genome copy numbers, which also act as viral polyprotein mRNA and are thus linked to viral gene expression, were measured by real time quantitative PCR targeting a region of nsP3. RNAs from three independent biological replicates (for both non-stimulated and stimulated cells) were reverse transcribed in triplicate; each reverse transcription reaction was then amplified in triplicate. Each bar represents the average of all measurements from a representative real time quantitative PCR experiment (see Experiemental procedures); error bars represent the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Article Snippet: Pathway stimulation with Escherichia coli To prepare working stocks of E. coli strain JM109 (New England Biolabs, Hitchim, UK), 1 µl of bacterial stock solution was added to 5 ml LB medium (without antibiotics), incubated at 37 °C/18 h, and centrifuged (4 °C, 2500 rpm/10 min).

    Techniques: Activation Assay, Radial Immuno Diffusion, Infection, Isolation, Activated Clotting Time Assay, Expressing, Real-time Polymerase Chain Reaction, Amplification, Standard Deviation

    Effects of host cell defence signaling on SFV gene expression. (A) Recombinant SFV4-derived viruses expressing Renilla luciferase ( Rluc ) from the non-structural (SFV4(3H)- Rluc ) or structural region of the genome (SFV4-st Rluc ). (B) U4.4 cells were stimulated with heat-inactivated Escherichia coli for 1 h or mock-treated, then infected (m.o.i. 1) with recombinant SFVs. Luciferase expression was measured at 12 h p.i. (C) U4.4 cells were transfected with constitutively active Toll ΔLRR receptor (pJL195) or mock-stimulated (transfection of pIB-V5/His; empty insect cell expression vector) for 24 h then infected with (m.o.i. 1) SFV4(3H)- Rluc or SFV4-st Rluc . Cells were lysed 12 h p.i. and luciferase activities determined. Each bar represents the mean of three independent biological replicates; error bars indicate the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Journal: Insect Molecular Biology

    Article Title: Semliki Forest virus strongly reduces mosquito host defence signaling

    doi: 10.1111/j.1365-2583.2008.00834.x

    Figure Lengend Snippet: Effects of host cell defence signaling on SFV gene expression. (A) Recombinant SFV4-derived viruses expressing Renilla luciferase ( Rluc ) from the non-structural (SFV4(3H)- Rluc ) or structural region of the genome (SFV4-st Rluc ). (B) U4.4 cells were stimulated with heat-inactivated Escherichia coli for 1 h or mock-treated, then infected (m.o.i. 1) with recombinant SFVs. Luciferase expression was measured at 12 h p.i. (C) U4.4 cells were transfected with constitutively active Toll ΔLRR receptor (pJL195) or mock-stimulated (transfection of pIB-V5/His; empty insect cell expression vector) for 24 h then infected with (m.o.i. 1) SFV4(3H)- Rluc or SFV4-st Rluc . Cells were lysed 12 h p.i. and luciferase activities determined. Each bar represents the mean of three independent biological replicates; error bars indicate the standard deviation. Every experiment was repeated at least twice under the same conditions.

    Article Snippet: Pathway stimulation with Escherichia coli To prepare working stocks of E. coli strain JM109 (New England Biolabs, Hitchim, UK), 1 µl of bacterial stock solution was added to 5 ml LB medium (without antibiotics), incubated at 37 °C/18 h, and centrifuged (4 °C, 2500 rpm/10 min).

    Techniques: Expressing, Recombinant, Derivative Assay, Luciferase, Infection, Transfection, Plasmid Preparation, Standard Deviation

    E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Journal: Nature Communications

    Article Title: Transcription factor YcjW controls the emergency H2S production in E. coli

    doi: 10.1038/s41467-019-10785-x

    Figure Lengend Snippet: E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H 2 S levels and tolerance to Gm and H 2 O 2 . a Δ mstA-sup has increased survival rate compared with Δ mstA when treated with 2 µg ml −1 gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b Δ mstA-sup also has increased tolerance after exposure to 5 mM H 2 O 2 for 30 min. c H 2 S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD 600 and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H 2 S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD ( n = 3). * p

    Article Snippet: To generate pLLY1, ycjW was PCR amplified from E. coli MG1655 using primers LL10 and LL11 and cloned into pACYC184 plasmid (NEB) using the Gibson Assembly Mastermix, according to the manufacturer’s protocol (NEB).

    Techniques: Time-Kill Assay, Staining

    ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Article Snippet: Validation of DNA Substrates Containing N7-meG and Me-FAPy-G. DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Fluorescence

    FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Article Snippet: Validation of DNA Substrates Containing N7-meG and Me-FAPy-G. DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence

    Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.

    Article Snippet: Validation of DNA Substrates Containing N7-meG and Me-FAPy-G. DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence