phi 29 dna polymerase  (Thermo Fisher)


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    Name:
    phi29 DNA Polymerase 10 U µL
    Description:
    Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase up to more than 70 kb featuring strong strand displacement activity which allows for highly efficient isothermal DNA amplification phi29 DNA Polymerase also possesses a 3 →5 exonuclease proofreading activity acting preferentially on single stranded DNA or RNA Therefore 3 modified primers are highly recommended Highlights• Highest processivity and strand displacement activity among known DNA polymerases more than 70 kb long DNA stretches can be synthesized• Highly accurate DNA synthesis• Extremely high yields of amplified DNA even from minute amounts of template• Amplification products can be directly used in downstream applications PCR restriction digestion SNP genotyping etc Applications• Rolling circle amplification RCA generation of periodic DNA nanotemplates• Multiple displacement amplification MDA • Unbiased amplification of whole genome WGA see Figure 1 in Supporting Data • amplification of DNA for SNP and STR detection• cell free amplification of DNA from single cells• pathogenic organisms or metagenomes• amplification of DNA from filter paper blood spot samples• DNA template preparation for sequencing• Protein primed DNA amplification• In situ genotyping with padlock probesRecombination based cloning• Cell free cloning of lethal DNA• RNA primed DNA amplificationNoteAddition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis Use of this enzyme in certain applications may be covered by patents and may require a license
    Catalog Number:
    ep0091
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher phi 29 dna polymerase
    Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using <t>phi</t> 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.
    Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase up to more than 70 kb featuring strong strand displacement activity which allows for highly efficient isothermal DNA amplification phi29 DNA Polymerase also possesses a 3 →5 exonuclease proofreading activity acting preferentially on single stranded DNA or RNA Therefore 3 modified primers are highly recommended Highlights• Highest processivity and strand displacement activity among known DNA polymerases more than 70 kb long DNA stretches can be synthesized• Highly accurate DNA synthesis• Extremely high yields of amplified DNA even from minute amounts of template• Amplification products can be directly used in downstream applications PCR restriction digestion SNP genotyping etc Applications• Rolling circle amplification RCA generation of periodic DNA nanotemplates• Multiple displacement amplification MDA • Unbiased amplification of whole genome WGA see Figure 1 in Supporting Data • amplification of DNA for SNP and STR detection• cell free amplification of DNA from single cells• pathogenic organisms or metagenomes• amplification of DNA from filter paper blood spot samples• DNA template preparation for sequencing• Protein primed DNA amplification• In situ genotyping with padlock probesRecombination based cloning• Cell free cloning of lethal DNA• RNA primed DNA amplificationNoteAddition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis Use of this enzyme in certain applications may be covered by patents and may require a license
    https://www.bioz.com/result/phi 29 dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phi 29 dna polymerase - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine"

    Article Title: A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine

    Journal: Microorganisms

    doi: 10.3390/microorganisms8081191

    Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.
    Figure Legend Snippet: Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.

    Techniques Used: Amplification

    Related Articles

    Amplification:

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. In this context, DNA amplification driven by phi29 DNA polymerase provides an alternative approach to amplify long DNA molecules and because of isothermal reaction conditions [ , ], the potential problems associated with emulsion stability become irrelevant. .. Moreover, the ability not only to amplify individual DNA molecules, but also to express genes from the amplified template opens many interesting possibilities for high throughput screening applications.

    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: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    Article Title: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas
    Article Snippet: .. ALT cells, but not telomerase positive cells, generate extra-chromosomal telomeric single-stranded DNA called C-circles, which can be amplified using the Phi29 DNA polymerase [ ] (Fig. ). ..

    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.

    Multiple Displacement Amplification:

    Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA
    Article Snippet: .. First, using the standard MDA protocol, we evaluated the purity of phi29 DNA polymerases from various vendors, including Lucigen (Middleton, WI), Epicentre (Madison, WI), Thermo Scientific (St. Louis, MO), and New England Biolabs (Ipswich, MA). .. Decontamination through UV irradiation as described by Woyke et al. was also adopted for the reagents, including phi29 DNA polymerase, dNTPs, and primers ( ).

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    Mutagenesis:

    Article Title: Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme
    Article Snippet: .. BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB. .. RNA labeling KinaseMax kit was purchased from Ambion; yeast tRNA was obtained from Serva.

    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.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    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.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

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    Thermo Fisher phi 29 dna polymerase
    Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using <t>phi</t> 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.
    Phi 29 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi 29 dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phi 29 dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.

    Journal: Microorganisms

    Article Title: A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine

    doi: 10.3390/microorganisms8081191

    Figure Lengend Snippet: Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.

    Article Snippet: One DNA extract of the beef sample enriched for 16 h and extracted with the Nucleospin Food kit was amplified using phi 29 DNA polymerase (ThermoFisher scientific, Waltham, MA, USA) according to the manufacturer’s instructions (method E, ).

    Techniques: Amplification