bsa  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Name:
    phi29 DNA Polymerase
    Description:
    phi29 DNA Polymerase 1 250 units
    Catalog Number:
    m0269l
    Price:
    228
    Size:
    1 250 units
    Category:
    DNA Polymerases
    Buy from Supplier


    Structured Review

    New England Biolabs bsa
    phi29 DNA Polymerase
    phi29 DNA Polymerase 1 250 units
    https://www.bioz.com/result/bsa/product/New England Biolabs
    Average 95 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    bsa - by Bioz Stars, 2020-02
    95/100 stars

    Images

    Related Articles

    Amplification:

    Article Title: Development of an easy and cost-effective method for non-invasive genotyping of insects
    Article Snippet: The COI region from M . separata frass DNA was amplified from all samples using Multiple Displacement Amplification (MDA), a non-PCR-based DNA amplification technique that can rapidly amplify minute amounts of DNA samples to a reasonable quantity by using the high fidelity, processivity and standard displacement capacity of phi 29 DNA polymerase [ ]. .. The MDA reaction was carried out in a 20 μl final volume containing 2 μl 10× reaction buffer with 1.6 μl dNTP mix (2.5 μM each), 0.5 μl phi29 DNA polymerase (0.5 U, New England Biolabs), 0.5 μl hexamer primer (10 μM), and DNA template (~50 ng).

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: .. The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively. .. Plasmid DNA isolation was checked via qPCR against a specific plasmid-borne gene on all three plasmids. qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) per the manufacturer’s protocol.

    Article Title: TZAP: a telomere-associated protein involved in telomere length control
    Article Snippet: The method for T-circle amplification was performed as described in ( ) with some modifications. .. The annealed sample was divided in two and half (10 μl) was incubated at 30°C for 12 h in the presence of dNTPs (200 μM each), 0.2 mg/ml BSA (NEB), 1X phi29 Buffer (NEB) and 7.5 U phi29 DNA polymerase (NEB) in a total volume of 20 μl.

    Article Title: Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA ▿
    Article Snippet: Paragraph title: Rolling circle amplification. ... Sample mixtures were combined with 10 μl of reaction mixture containing: 1× Phi29 buffer, the eight primers at a concentration of 0.5 μM each, 0.4 mg/ml of bovine serum albumin, 4 mM of dithiothreitol, 2 mM of each dNTP, and 10 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA).

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: .. The denatured DNA was amplified using 0.3 μL Phi29 DNA polymerase (10 units/μL, New England Biolabs) complemented with 2 μL of 10 × Phi29 DNA polymerase buffer, 0.2μL of 100 × BSA, 3.2μL of 2.5mM dNTP (Applied Biosystems), 1 μL of 20% DMSO (Sigma), and 1 μL of 20 ng/μL T4 gene32 (Amersham Biosciences) in a volume of 20 μL at 30°C for 16 h. The Phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was digested with 2.5 μL of Nla-III at 37°C for 3 h in a volume of 100 μL. .. MDA was performed for target (BT474) and reference genomic DNAs using the Repli-g whole genome amplification kit (Molecular Staging) according to kit instructions.

    Article Title: Characterization of a novel gyrovirus in human stool and chicken meat
    Article Snippet: .. To sequence the complete genome of the putative novel gyrovirus, nucleic acid extracted from the original sample was subjected to rolling circle DNA amplification using Phi29 DNA polymerase (New England BioLabs, Massachusetts, USA). .. Inverse PCR was performed on the rolling circle amplified viral DNA using Platinum Pfx DNA polymerase (Invitrogen, California, USA).

    Article Title: A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR
    Article Snippet: .. In order to increase the efficiency of amplification, RCA was divided into two steps: First-step, 14 μl primer combining reaction mixture containing primers at a operative solutionconcentration of 0.5 μmol/l each , 2 μl reaction buffer and 11 μl ddH2 O were loaded onto the tissue sections on glass slides and sealed with rubber cement, followed by heating at 95°C for 6 min, and then placed on ice for 15 min. Second-step the RCA amplification was as follows: 23 μl of reaction mixture was added to tissue slides containing primers at a operative solution concentration of 0.5 μmol/l each, 10 mg/ml bovine serum albumin (BSA), 0.32 mmol/l of dNTP, 20 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA), and 2 μl reaction buffer. .. Then Phi29 DNA polymerase was inactivated at 70°C for10 min.

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: .. Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment. .. The RCA reaction was incubated at 37 °C for 1 hour, followed by 65 °C for 10 minutes.

    Whole Genome Amplification:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Paragraph title: RCA–RCA whole genome amplification ... The denatured DNA was amplified using 0.3 μL Phi29 DNA polymerase (10 units/μL, New England Biolabs) complemented with 2 μL of 10 × Phi29 DNA polymerase buffer, 0.2μL of 100 × BSA, 3.2μL of 2.5mM dNTP (Applied Biosystems), 1 μL of 20% DMSO (Sigma), and 1 μL of 20 ng/μL T4 gene32 (Amersham Biosciences) in a volume of 20 μL at 30°C for 16 h. The Phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was digested with 2.5 μL of Nla-III at 37°C for 3 h in a volume of 100 μL.

    Article Title: Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations
    Article Snippet: Paragraph title: Selective whole genome amplification ... SWGA reactions were performed containing a maximum of 50 ng of total input genomic DNA (and a minimum of 5 ng), 5 µl of 10 x Phi29 DNA Polymerase Reaction Buffer (New England BioLabs), 0.5 µl of Purified 100x BSA (New England BioLabs), 0.5 µl of 250 µM Primer mix of Pkset1, 5 µl 10 mM dNTP (Roche), 30 units Phi29 DNA Polymerase (New England BioLabs) and Nuclease-Free Water (Ambion, The RNA Company) to reach a final reaction volume of 50 µl.

    Synthesized:

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
    Article Snippet: Synthetic oligonucleotides and miRNAs were synthesized and purified with polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies (IDT, Coralville, IA). .. Phi29 DNA polymerase, T4 DNA ligase, Nb.BbvCI nicking endonuclease and deoxynucleotide solution mixture (dNTPs) were purchased from New England Biolabs.

    SYBR Green Assay:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively. .. Plasmid DNA isolation was checked via qPCR against a specific plasmid-borne gene on all three plasmids. qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) per the manufacturer’s protocol.

    Article Title: Repressor logic modules assembled by rolling circle amplification platform to construct a set of logic gates
    Article Snippet: .. Circularization and RCA reaction For circularization, The 5′- and 3′-portions of the circular template (1 μM) were hybridized with a splint oligonucleotide (1 μM) in a head-to-tail way, and then were ligated by T4 DNA ligase (175 U) in ligation buffer containing 50 mM Tris pH 8.0, 10 mM MgCl2 , 5 mM DTT and 0.1 mM ATP at 16 °C for 60 min. RCA reaction solution was composed of 0.05 pmol ligated circular template, 50 mM Tris, 10 mM MgCl2 , 10 mM (NH4 )2 SO4 , 4 mM DTT (pH 7.5), SYBR Green II (1:10000) and phi29 DNA polymerase 3 U in 100 μL. ..

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
    Article Snippet: Phi29 DNA polymerase, T4 DNA ligase, Nb.BbvCI nicking endonuclease and deoxynucleotide solution mixture (dNTPs) were purchased from New England Biolabs. .. SYBR Green I, 2propanol (IPA, 99.5%), bovine serum albumin (BSA), aminopropyl-trimethoxysilane (APTMS), glutaraldehyde (GL, 50%), trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (97%), 3-glycidoxypropyltrimethoxysilane (GOPS, 97%) and triethylamine (TEA) were purchased from Sigma-Aldrich.

    Incubation:

    Article Title: Development of an easy and cost-effective method for non-invasive genotyping of insects
    Article Snippet: The MDA reaction was carried out in a 20 μl final volume containing 2 μl 10× reaction buffer with 1.6 μl dNTP mix (2.5 μM each), 0.5 μl phi29 DNA polymerase (0.5 U, New England Biolabs), 0.5 μl hexamer primer (10 μM), and DNA template (~50 ng). .. These samples were incubated at 30°C for 3–4 h followed by deactivation of the enzymes by heating to 65°C for 12 min. After deactivation, the product was purified through an AxyGen PCR purification kit, diluted finally with 20 μl eluent water and stored at -20°C for future use.

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: .. Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube. .. The reaction mixtures were incubated at 30°C for 6 to 8 h. The MDA product was purified using Zymo DNA clean-and-concentrator columns and prepared for sequencing using a Nextera DNA Library Prep kit.

    Article Title: TZAP: a telomere-associated protein involved in telomere length control
    Article Snippet: .. The annealed sample was divided in two and half (10 μl) was incubated at 30°C for 12 h in the presence of dNTPs (200 μM each), 0.2 mg/ml BSA (NEB), 1X phi29 Buffer (NEB) and 7.5 U phi29 DNA polymerase (NEB) in a total volume of 20 μl. .. The remaining half was incubated under the same conditions but excluding phi29 DNA polymerase from the reaction.

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment. .. The RCA reaction was incubated at 37 °C for 1 hour, followed by 65 °C for 10 minutes.

    Activity Assay:

    Article Title: Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA ▿
    Article Snippet: To protect primers from the strong proofreading activity of the Phi29 polymerase, the two 3′ bases were phosphorothioate modified. .. Sample mixtures were combined with 10 μl of reaction mixture containing: 1× Phi29 buffer, the eight primers at a concentration of 0.5 μM each, 0.4 mg/ml of bovine serum albumin, 4 mM of dithiothreitol, 2 mM of each dNTP, and 10 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA).

    Modification:

    Article Title: Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA ▿
    Article Snippet: To protect primers from the strong proofreading activity of the Phi29 polymerase, the two 3′ bases were phosphorothioate modified. .. Sample mixtures were combined with 10 μl of reaction mixture containing: 1× Phi29 buffer, the eight primers at a concentration of 0.5 μM each, 0.4 mg/ml of bovine serum albumin, 4 mM of dithiothreitol, 2 mM of each dNTP, and 10 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA).

    Filtration:

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube. .. RNA samples from extracellular fractions (with and without filtration using 0.45-µm-pore-size filters and with and without DNase treatment) were sequenced using only the SMARTer Stranded and MDA methods as there was not enough input material for the small-RNA sequencing method described in reference .

    Hybridization:

    Article Title: TZAP: a telomere-associated protein involved in telomere length control
    Article Snippet: The annealed sample was divided in two and half (10 μl) was incubated at 30°C for 12 h in the presence of dNTPs (200 μM each), 0.2 mg/ml BSA (NEB), 1X phi29 Buffer (NEB) and 7.5 U phi29 DNA polymerase (NEB) in a total volume of 20 μl. .. T-circle products were visualized by in-gel hybridization using a P32 end-labeled (TTAGGG)4 oligonucleotide probe.

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: Following hybridization and ligation, 39 µl of an exonuclease master mix consisting of 20 U Exonuclease I (New England Biolabs), 5 U Lambda exonuclease (New England Biolabs), 100 U Exonuclease III (New England Biolabs), 10 U Uracil dehydrogenase (New England Biolabs) and 28% Tween20 was added to each reaction. .. Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment.

    Inverse PCR:

    Article Title: Characterization of a novel gyrovirus in human stool and chicken meat
    Article Snippet: To sequence the complete genome of the putative novel gyrovirus, nucleic acid extracted from the original sample was subjected to rolling circle DNA amplification using Phi29 DNA polymerase (New England BioLabs, Massachusetts, USA). .. Inverse PCR was performed on the rolling circle amplified viral DNA using Platinum Pfx DNA polymerase (Invitrogen, California, USA).

    Ligation:

    Article Title: Repressor logic modules assembled by rolling circle amplification platform to construct a set of logic gates
    Article Snippet: .. Circularization and RCA reaction For circularization, The 5′- and 3′-portions of the circular template (1 μM) were hybridized with a splint oligonucleotide (1 μM) in a head-to-tail way, and then were ligated by T4 DNA ligase (175 U) in ligation buffer containing 50 mM Tris pH 8.0, 10 mM MgCl2 , 5 mM DTT and 0.1 mM ATP at 16 °C for 60 min. RCA reaction solution was composed of 0.05 pmol ligated circular template, 50 mM Tris, 10 mM MgCl2 , 10 mM (NH4 )2 SO4 , 4 mM DTT (pH 7.5), SYBR Green II (1:10000) and phi29 DNA polymerase 3 U in 100 μL. ..

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: Following hybridization and ligation, 39 µl of an exonuclease master mix consisting of 20 U Exonuclease I (New England Biolabs), 5 U Lambda exonuclease (New England Biolabs), 100 U Exonuclease III (New England Biolabs), 10 U Uracil dehydrogenase (New England Biolabs) and 28% Tween20 was added to each reaction. .. Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment.

    Indirect Immunoperoxidase Assay:

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
    Article Snippet: Phi29 DNA polymerase, T4 DNA ligase, Nb.BbvCI nicking endonuclease and deoxynucleotide solution mixture (dNTPs) were purchased from New England Biolabs. .. SYBR Green I, 2propanol (IPA, 99.5%), bovine serum albumin (BSA), aminopropyl-trimethoxysilane (APTMS), glutaraldehyde (GL, 50%), trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (97%), 3-glycidoxypropyltrimethoxysilane (GOPS, 97%) and triethylamine (TEA) were purchased from Sigma-Aldrich.

    other:

    Article Title: LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro
    Article Snippet: Nicking 5′ of the reactive deoxyadenosine within one of the integrated substrate molecules prior to the treatment with phi29 DNA polymerase could explain their occurrence.

    Sequencing:

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: The first was described previously ( ); this method provides unbiased sequencing, especially of shorter RNA fragments that may be missed by other protocols. .. Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube.

    Article Title: Characterization of a novel gyrovirus in human stool and chicken meat
    Article Snippet: .. To sequence the complete genome of the putative novel gyrovirus, nucleic acid extracted from the original sample was subjected to rolling circle DNA amplification using Phi29 DNA polymerase (New England BioLabs, Massachusetts, USA). .. Inverse PCR was performed on the rolling circle amplified viral DNA using Platinum Pfx DNA polymerase (Invitrogen, California, USA).

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: .. Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment. .. The RCA reaction was incubated at 37 °C for 1 hour, followed by 65 °C for 10 minutes.

    Binding Assay:

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: Four microliters circular DNA was mixed with 0.5 μL hexamers (400 ng/μL, Sigma) and 0.5 μL binding buffer (400 mM Tris-HCl at pH 8.0, 160 mM KCl) and denatured at 95°C for 4 min. Alternatively, the denaturation step was omitted. .. The denatured DNA was amplified using 0.3 μL Phi29 DNA polymerase (10 units/μL, New England Biolabs) complemented with 2 μL of 10 × Phi29 DNA polymerase buffer, 0.2μL of 100 × BSA, 3.2μL of 2.5mM dNTP (Applied Biosystems), 1 μL of 20% DMSO (Sigma), and 1 μL of 20 ng/μL T4 gene32 (Amersham Biosciences) in a volume of 20 μL at 30°C for 16 h. The Phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was digested with 2.5 μL of Nla-III at 37°C for 3 h in a volume of 100 μL.

    DNA Extraction:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: Paragraph title: Plasmid DNA isolation optimization. ... The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively.

    Marker:

    Article Title: Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
    Article Snippet: T4 DNA ligase, Phi29 DNA polymerase, nicking endonuclease Nb.BbvCI, deoxyribonucleoside 5′-triphosphate mixture (dNTPs) and exonuclease I (Exo I) were obtained from New England Biolabs (Beijing, China). .. Agarose, ethidium bromide (EB), ammonium persulfate, DNA Marker IV and D2000 were obtained from Tiangen Biotech.

    Article Title: Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures
    Article Snippet: DNA marker, T4 DNA ligase, Exonuclease I, Exonuclease III, RiboLock RNase Inhibitor and Trizol Reagent were obtained from Takara Biotechnology Co. Ltd (Dalian, China). .. Poly(U) polymerase, phi29 DNA polymerase and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs Ltd (Beijing, China) and RNase I from ThermoFisher Scientific.

    RNA Sequencing Assay:

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: Paragraph title: RNA sequencing of Spirulina samples. ... Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube.

    Multiple Displacement Amplification:

    Article Title: Development of an easy and cost-effective method for non-invasive genotyping of insects
    Article Snippet: .. The MDA reaction was carried out in a 20 μl final volume containing 2 μl 10× reaction buffer with 1.6 μl dNTP mix (2.5 μM each), 0.5 μl phi29 DNA polymerase (0.5 U, New England Biolabs), 0.5 μl hexamer primer (10 μM), and DNA template (~50 ng). .. These samples were incubated at 30°C for 3–4 h followed by deactivation of the enzymes by heating to 65°C for 12 min. After deactivation, the product was purified through an AxyGen PCR purification kit, diluted finally with 20 μl eluent water and stored at -20°C for future use.

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: .. Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube. .. The reaction mixtures were incubated at 30°C for 6 to 8 h. The MDA product was purified using Zymo DNA clean-and-concentrator columns and prepared for sequencing using a Nextera DNA Library Prep kit.

    Labeling:

    Article Title: Imaging single DNA molecules for high precision NIPT
    Article Snippet: Rolling circle amplification (RCA) was performed by adding 11.2 µl RCA master mix consisting of 1.07 µM of a RCA primer oligonucleotide (IDT) designed to be complementary to a sequence present in the backbone region of each DNA circle, 5.4 mM dNTP solution mix (New England Biolabs) and 20 U Phi29 DNA polymerase (New England Biolabs) to each reaction following exonuclease treatment. .. Following RCA a 12 µl labeling master mix was added to each reaction consisting of 60 nM of each fluorescent labeling oligonucleotide complementary to a chromosomal tag in the backbone, 12 × SSC buffer and 0.6% Tween20.

    Purification:

    Article Title: Development of an easy and cost-effective method for non-invasive genotyping of insects
    Article Snippet: The MDA reaction was carried out in a 20 μl final volume containing 2 μl 10× reaction buffer with 1.6 μl dNTP mix (2.5 μM each), 0.5 μl phi29 DNA polymerase (0.5 U, New England Biolabs), 0.5 μl hexamer primer (10 μM), and DNA template (~50 ng). .. These samples were incubated at 30°C for 3–4 h followed by deactivation of the enzymes by heating to 65°C for 12 min. After deactivation, the product was purified through an AxyGen PCR purification kit, diluted finally with 20 μl eluent water and stored at -20°C for future use.

    Article Title: On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
    Article Snippet: Subsequently, 127.5 μl of multiple-displacement amplification (MDA) master mix (6 μl of 25 mM dNTP mix [Roche], 15 μl of 10x Phi29 DNA polymerase reaction buffer [NEB], 7.5 μl of 1 μg/μl exonuclease-resistant random hexamers, 7.5 μl of 0.1 M dithiothreitol, 93 μl of RNase-free water) was added to each reaction, and the mixture was split into 3 tubes at 47.5 μl each and incubated at 95°C for 5 min and then at 4°C during the addition of 2.5 μl of Phi29 DNA polymerase (NEB) to each tube. .. The reaction mixtures were incubated at 30°C for 6 to 8 h. The MDA product was purified using Zymo DNA clean-and-concentrator columns and prepared for sequencing using a Nextera DNA Library Prep kit.

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: The fragmented DNAs were circularized with 0.5 μL T4 DNA ligase (2000 units/μL, New England Biolabs) in a volume of 15 μL at room temperature for 2 h. After inactivation of ligase at 65°C for 10 min, linear DNAs were eliminated with 1.2 μL Lamda Exonuclease (5 units/μL, New England Biolabs) and 0.3 μL Exonuclease I (20 units/μL, New England Biolabs) in a volume of 25 μL at 37°C for 1 h. The circularized DNAs were then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 35 μL of H2 O. .. The denatured DNA was amplified using 0.3 μL Phi29 DNA polymerase (10 units/μL, New England Biolabs) complemented with 2 μL of 10 × Phi29 DNA polymerase buffer, 0.2μL of 100 × BSA, 3.2μL of 2.5mM dNTP (Applied Biosystems), 1 μL of 20% DMSO (Sigma), and 1 μL of 20 ng/μL T4 gene32 (Amersham Biosciences) in a volume of 20 μL at 30°C for 16 h. The Phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was digested with 2.5 μL of Nla-III at 37°C for 3 h in a volume of 100 μL.

    Article Title: Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations
    Article Snippet: .. SWGA reactions were performed containing a maximum of 50 ng of total input genomic DNA (and a minimum of 5 ng), 5 µl of 10 x Phi29 DNA Polymerase Reaction Buffer (New England BioLabs), 0.5 µl of Purified 100x BSA (New England BioLabs), 0.5 µl of 250 µM Primer mix of Pkset1, 5 µl 10 mM dNTP (Roche), 30 units Phi29 DNA Polymerase (New England BioLabs) and Nuclease-Free Water (Ambion, The RNA Company) to reach a final reaction volume of 50 µl. ..

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
    Article Snippet: Synthetic oligonucleotides and miRNAs were synthesized and purified with polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies (IDT, Coralville, IA). .. Phi29 DNA polymerase, T4 DNA ligase, Nb.BbvCI nicking endonuclease and deoxynucleotide solution mixture (dNTPs) were purchased from New England Biolabs.

    Polymerase Chain Reaction:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: If 16S rRNA PCR product was visible on a 1% agarose gel, another overnight digestion reaction was performed until the product could no longer be visualized. .. The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively.

    Article Title: DNA amplification method tolerant to sample degradation
    Article Snippet: The fragmented DNAs were circularized with 0.5 μL T4 DNA ligase (2000 units/μL, New England Biolabs) in a volume of 15 μL at room temperature for 2 h. After inactivation of ligase at 65°C for 10 min, linear DNAs were eliminated with 1.2 μL Lamda Exonuclease (5 units/μL, New England Biolabs) and 0.3 μL Exonuclease I (20 units/μL, New England Biolabs) in a volume of 25 μL at 37°C for 1 h. The circularized DNAs were then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 35 μL of H2 O. .. The denatured DNA was amplified using 0.3 μL Phi29 DNA polymerase (10 units/μL, New England Biolabs) complemented with 2 μL of 10 × Phi29 DNA polymerase buffer, 0.2μL of 100 × BSA, 3.2μL of 2.5mM dNTP (Applied Biosystems), 1 μL of 20% DMSO (Sigma), and 1 μL of 20 ng/μL T4 gene32 (Amersham Biosciences) in a volume of 20 μL at 30°C for 16 h. The Phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was digested with 2.5 μL of Nla-III at 37°C for 3 h in a volume of 100 μL.

    Article Title: Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations
    Article Snippet: All SWGA reactions were carried out in the UV Cabinet for PCR Operations (UV-B-AR, Grant-Bio) to minimize contamination. .. SWGA reactions were performed containing a maximum of 50 ng of total input genomic DNA (and a minimum of 5 ng), 5 µl of 10 x Phi29 DNA Polymerase Reaction Buffer (New England BioLabs), 0.5 µl of Purified 100x BSA (New England BioLabs), 0.5 µl of 250 µM Primer mix of Pkset1, 5 µl 10 mM dNTP (Roche), 30 units Phi29 DNA Polymerase (New England BioLabs) and Nuclease-Free Water (Ambion, The RNA Company) to reach a final reaction volume of 50 µl.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
    Article Snippet: Synthetic oligonucleotides and miRNAs were synthesized and purified with polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies (IDT, Coralville, IA). .. Phi29 DNA polymerase, T4 DNA ligase, Nb.BbvCI nicking endonuclease and deoxynucleotide solution mixture (dNTPs) were purchased from New England Biolabs.

    Plasmid Preparation:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: Paragraph title: Plasmid DNA isolation optimization. ... The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively.

    Software:

    Article Title: Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA
    Article Snippet: Molar extinction coefficient was determined by OligoAnayzer 3.1 software provided in the following website ( http://sg.idtdna.com/calc/analyzer ). .. T4 DNA ligase, Phi29 DNA polymerase, nicking endonuclease Nb.BbvCI, deoxyribonucleoside 5′-triphosphate mixture (dNTPs) and exonuclease I (Exo I) were obtained from New England Biolabs (Beijing, China).

    Real-time Polymerase Chain Reaction:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively. .. Plasmid DNA isolation was checked via qPCR against a specific plasmid-borne gene on all three plasmids. qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) per the manufacturer’s protocol.

    Agarose Gel Electrophoresis:

    Article Title: Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes
    Article Snippet: If 16S rRNA PCR product was visible on a 1% agarose gel, another overnight digestion reaction was performed until the product could no longer be visualized. .. The DNA was then amplified with Phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) ( ) at 4, 18, or 30°C for 168, 25, and 24 h, respectively.

    Next-Generation Sequencing:

    Article Title: Characterization of a novel gyrovirus in human stool and chicken meat
    Article Snippet: Roche 454 high throughput sequencing was applied to nucleic acid extracted individually from 96 faecal samples from patients with diarrhoea from whom no other viral pathogen had been identified. .. To sequence the complete genome of the putative novel gyrovirus, nucleic acid extracted from the original sample was subjected to rolling circle DNA amplification using Phi29 DNA polymerase (New England BioLabs, Massachusetts, USA).

    Concentration Assay:

    Article Title: Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA ▿
    Article Snippet: .. Sample mixtures were combined with 10 μl of reaction mixture containing: 1× Phi29 buffer, the eight primers at a concentration of 0.5 μM each, 0.4 mg/ml of bovine serum albumin, 4 mM of dithiothreitol, 2 mM of each dNTP, and 10 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA). .. First, 2 μl of RCA products was run on an 0.8% agarose gel and stained with ethidium bromide.

    Article Title: A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR
    Article Snippet: .. In order to increase the efficiency of amplification, RCA was divided into two steps: First-step, 14 μl primer combining reaction mixture containing primers at a operative solutionconcentration of 0.5 μmol/l each , 2 μl reaction buffer and 11 μl ddH2 O were loaded onto the tissue sections on glass slides and sealed with rubber cement, followed by heating at 95°C for 6 min, and then placed on ice for 15 min. Second-step the RCA amplification was as follows: 23 μl of reaction mixture was added to tissue slides containing primers at a operative solution concentration of 0.5 μmol/l each, 10 mg/ml bovine serum albumin (BSA), 0.32 mmol/l of dNTP, 20 U of the Phi29 DNA polymerase (New England Biolabs, Worcester, MA), and 2 μl reaction buffer. .. Then Phi29 DNA polymerase was inactivated at 70°C for10 min.

    High Throughput Screening Assay:

    Article Title: Characterization of a novel gyrovirus in human stool and chicken meat
    Article Snippet: Paragraph title: Viral genetic detection and characterization by high-throughput deep-sequencing methods ... To sequence the complete genome of the putative novel gyrovirus, nucleic acid extracted from the original sample was subjected to rolling circle DNA amplification using Phi29 DNA polymerase (New England BioLabs, Massachusetts, USA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    New England Biolabs phi 29 polymerase
    Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a <t>phi-29</t> amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.
    Phi 29 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi 29 polymerase/product/New England Biolabs
    Average 90 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    phi 29 polymerase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    Image Search Results


    Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Plasmid Preparation, Isolation, Clone Assay, Transformation Assay, Amplification, Marker, Functional Assay

    (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Polymerase Chain Reaction, Clone Assay, Random Primed, Amplification, Agarose Gel Electrophoresis, Diagnostic Assay, Plasmid Preparation

    ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Southern Blot, Sequencing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Diagnostic Assay, Amplification, BAC Assay

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Electroporation, Amplification

    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Journal: Nucleic Acids Research

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    doi: 10.1093/nar/gkl350

    Figure Lengend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Journal: Nucleic Acids Research

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    doi: 10.1093/nar/gkl350

    Figure Lengend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: Molecular Weight

    Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Journal: mBio

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    doi: 10.1128/mBio.02257-16

    Figure Lengend Snippet: Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Article Snippet: Thirty to 70 ng of input DNA was added to a 50-µl reaction mixture containing 3.5 µM SWGA primers, 30 U phi29 DNA polymerase enzyme (New England Biolabs), 1× phi29 buffer (New England Biolabs), 4 mM dNTPs (Roche), 1% bovine serum albumin, and water.

    Techniques: Whole Genome Amplification, Synthesized, Binding Assay, Amplification