t4 gene 32  (New England Biolabs)


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  • 99
    Name:
    T4 Gene 32 Protein
    Description:
    T4 Gene 32 Protein 500 ug
    Catalog Number:
    m0300l
    Price:
    318
    Size:
    500 ug
    Category:
    DNA Binding Proteins
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    Structured Review

    New England Biolabs t4 gene 32
    T4 Gene 32 Protein
    T4 Gene 32 Protein 500 ug
    https://www.bioz.com/result/t4 gene 32/product/New England Biolabs
    Average 99 stars, based on 23542 article reviews
    Price from $9.99 to $1999.99
    t4 gene 32 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA"

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku737

    RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.
    Figure Legend Snippet: RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.

    Techniques Used: Amplification, Sequencing, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis

    RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.
    Figure Legend Snippet: RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Negative Control, Picogreen Assay, Amplification, Fluorescence

    Single and double-stranded DNA production changes in RCA of p378 circular DNA over time. ( A ) BseGI digested RCA products performed without SSB protein over time (1–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( B ) BseGI digested RCA products performed with SSB protein over time (3–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( C ) Plotted concentrations of single and double-stranded RCA products expressed in nanograms per microliter over time. Single-stranded DNA was measured as deduction of the double-stranded DNA band intensities (agarose gels) from the total DNA band intensities (denaturing polyacrilamide gels). Linearity of the band intensities corresponding to the DNA amount range used for our experimental condition was verified (See Supplementary Figure S4). ( D ) Single and double-stranded DNA in RCA over time expressed as mass fraction. In the initial hours of a rolling circle amplification without SSB protein most of the amplicons are in the single-stranded form but then all the DNA is converted into the double-stranded form. The addition of SSB protein T4 gene 32 drastically reduces the conversion of single-stranded DNA into double-stranded form.
    Figure Legend Snippet: Single and double-stranded DNA production changes in RCA of p378 circular DNA over time. ( A ) BseGI digested RCA products performed without SSB protein over time (1–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( B ) BseGI digested RCA products performed with SSB protein over time (3–72 h) loaded on agarose gel (above) and denaturing polyacrilamide gel (below). ( C ) Plotted concentrations of single and double-stranded RCA products expressed in nanograms per microliter over time. Single-stranded DNA was measured as deduction of the double-stranded DNA band intensities (agarose gels) from the total DNA band intensities (denaturing polyacrilamide gels). Linearity of the band intensities corresponding to the DNA amount range used for our experimental condition was verified (See Supplementary Figure S4). ( D ) Single and double-stranded DNA in RCA over time expressed as mass fraction. In the initial hours of a rolling circle amplification without SSB protein most of the amplicons are in the single-stranded form but then all the DNA is converted into the double-stranded form. The addition of SSB protein T4 gene 32 drastically reduces the conversion of single-stranded DNA into double-stranded form.

    Techniques Used: Agarose Gel Electrophoresis, Amplification

    2) Product Images from "PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism"

    Article Title: PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh012

    Electrophoretic comparison of fragments 179 (lanes 1–6) and 109 (lanes 7–12) amplified by PCR from toxic and non-toxic strains of A.circinalis . Gel lanes 1–12 as follows: 131C (1 and 7), 344B (2 and 8), 134C (3 and 9), 279B (4 and 10), 306A (5 and 11) and 271C (6 and 12). PCR products were loaded as a total of 4 µl per sample and run with 1 kb Plus DNA Ladder (Invitrogen) as standard (lanes M).
    Figure Legend Snippet: Electrophoretic comparison of fragments 179 (lanes 1–6) and 109 (lanes 7–12) amplified by PCR from toxic and non-toxic strains of A.circinalis . Gel lanes 1–12 as follows: 131C (1 and 7), 344B (2 and 8), 134C (3 and 9), 279B (4 and 10), 306A (5 and 11) and 271C (6 and 12). PCR products were loaded as a total of 4 µl per sample and run with 1 kb Plus DNA Ladder (Invitrogen) as standard (lanes M).

    Techniques Used: Amplification, Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. We circularized the linear 378-nt pseudogene (5 ng/μl) by T4 ligase (0.25 U/μl; Fermentas) in 1× rapid ligation buffer at 22°C for 10 min, followed by an inactivation step at 65°C for 10 min. We nicked the resulting circular pseudogene (1 ng/μl) by Nb.BsrDI and Nt.BspQI (0.5 U/μl, New England Biolabs) in 1× NEB3 buffer at 65°C for 2 h and we stopped the reaction by heating at 80°C for 20 min. We amplified the nicked circular pseudogene (0.1 ng/μl) at 30°C by RCA and T4 gene 32 (25 ng/μl; NEB) stopping the reaction by heat inactivation (80°C, for 20 min) at different time points. .. We digested RCA products by BseGI restriction enzyme (0.75 U/μl; Fermentas), which recognizes GGATG(2/0)∧ sites, incubating in Tango 1× buffer for 24 h at 55°C.

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Each reaction component for C1-RamDA-seq was as follows: Lysis Final Mix (1.12 μL of 10% NP40, 4.05 μL RealTime ready Cell Lysis Buffer, 0.84 μL of 40 U/μL RNasin Plus RNase Inhibitor, 3 μL of 1:5000 ERCC RNA Spikes, 1.35 μL of C1 Loading Reagent, and 16.55 μL of RNase-free water), gDNA Digestion Final Mix (2.5 μL of 5× PrimeScript Buffer, 5 μL of 1 U/μL DNase I Amplification Grade, 1 μL of 20× C1 Loading Reagent, and 11.5 μL of RNase-free water), Priming Final Mix (17.23 μL of 5× PrimeScript Buffer, 5.24 μL of PrimeScript RT Enzyme Mix, 0.7 μL of 30 μM oligo(dT)12, 2.8 μL of 100 μM 1st-NSRs, 1.75 μL of 2 mg/mL T4 gene 32 protein (NEB), 1.13 μL of C1 Loading Reagent, and 1.15 μL of RNase-free water), RT Final Mix (12 μL of 5× PrimeScript Buffer, 3 μL of PrimeScript RT Enzyme Mix, RealTime ready Cell Lysis Buffer, 0.4 μL of 30 μM oligo(dT)12, 1.6 μL of 100 μM 1st-NSRs, 1 μL of 2 mg/ml T4 gene 32 protein (NEB), 3.96 μL of 1 U/μL DNase I Amplification Grade, 2.25 μL of C1 Loading Reagent, and 33.92 μL of RNase-free water), Second-strand Final Mix (6.7 μL of 10× NEB buffer 2, 6.7 μL of 2.5 mM each dNTP Mixture, 5.36 μL of 100 μM 2nd-NSRs, 2.01 μL of 5 U/μL Klenow Fragment (3′ → 5′ exo-), 1.5 μL of C1 Loading Reagent, and 7.73 μL of RNase-free water), and Harvest Reagent (500 μL of Tagment DNA Buffer, 237.5 μL of C1 Harvest Reagent, and 12.5 μL of 20× C1 Loading Reagent). .. For the 10-pg total RNA sample, 30 ng of total RNA was added to the Lysis Final Mix, and the Cell Wash Buffer (Fluidigm) was loaded for IFC rather than Cell Mix.

    Ligation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. We circularized the linear 378-nt pseudogene (5 ng/μl) by T4 ligase (0.25 U/μl; Fermentas) in 1× rapid ligation buffer at 22°C for 10 min, followed by an inactivation step at 65°C for 10 min. We nicked the resulting circular pseudogene (1 ng/μl) by Nb.BsrDI and Nt.BspQI (0.5 U/μl, New England Biolabs) in 1× NEB3 buffer at 65°C for 2 h and we stopped the reaction by heating at 80°C for 20 min. We amplified the nicked circular pseudogene (0.1 ng/μl) at 30°C by RCA and T4 gene 32 (25 ng/μl; NEB) stopping the reaction by heat inactivation (80°C, for 20 min) at different time points. .. We digested RCA products by BseGI restriction enzyme (0.75 U/μl; Fermentas), which recognizes GGATG(2/0)∧ sites, incubating in Tango 1× buffer for 24 h at 55°C.

    Concentration Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Incubation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Lysis:

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Each reaction component for C1-RamDA-seq was as follows: Lysis Final Mix (1.12 μL of 10% NP40, 4.05 μL RealTime ready Cell Lysis Buffer, 0.84 μL of 40 U/μL RNasin Plus RNase Inhibitor, 3 μL of 1:5000 ERCC RNA Spikes, 1.35 μL of C1 Loading Reagent, and 16.55 μL of RNase-free water), gDNA Digestion Final Mix (2.5 μL of 5× PrimeScript Buffer, 5 μL of 1 U/μL DNase I Amplification Grade, 1 μL of 20× C1 Loading Reagent, and 11.5 μL of RNase-free water), Priming Final Mix (17.23 μL of 5× PrimeScript Buffer, 5.24 μL of PrimeScript RT Enzyme Mix, 0.7 μL of 30 μM oligo(dT)12, 2.8 μL of 100 μM 1st-NSRs, 1.75 μL of 2 mg/mL T4 gene 32 protein (NEB), 1.13 μL of C1 Loading Reagent, and 1.15 μL of RNase-free water), RT Final Mix (12 μL of 5× PrimeScript Buffer, 3 μL of PrimeScript RT Enzyme Mix, RealTime ready Cell Lysis Buffer, 0.4 μL of 30 μM oligo(dT)12, 1.6 μL of 100 μM 1st-NSRs, 1 μL of 2 mg/ml T4 gene 32 protein (NEB), 3.96 μL of 1 U/μL DNase I Amplification Grade, 2.25 μL of C1 Loading Reagent, and 33.92 μL of RNase-free water), Second-strand Final Mix (6.7 μL of 10× NEB buffer 2, 6.7 μL of 2.5 mM each dNTP Mixture, 5.36 μL of 100 μM 2nd-NSRs, 2.01 μL of 5 U/μL Klenow Fragment (3′ → 5′ exo-), 1.5 μL of C1 Loading Reagent, and 7.73 μL of RNase-free water), and Harvest Reagent (500 μL of Tagment DNA Buffer, 237.5 μL of C1 Harvest Reagent, and 12.5 μL of 20× C1 Loading Reagent). .. For the 10-pg total RNA sample, 30 ng of total RNA was added to the Lysis Final Mix, and the Cell Wash Buffer (Fluidigm) was loaded for IFC rather than Cell Mix.

    Binding Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Plasmid Preparation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

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    New England Biolabs restriction buffer neb3 1
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    https://www.bioz.com/result/restriction buffer neb3 1/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction buffer neb3 1 - by Bioz Stars, 2020-09
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