phi29 dna polymerase buffer  (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 phi29 dna polymerase buffer
    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/phi29 dna polymerase buffer/product/Thermo Fisher
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase buffer - by Bioz Stars, 2020-11
    99/100 stars

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    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: 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. ). ..

    Southern Blot:

    Article Title: A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome
    Article Snippet: .. DNA was double digested by AluI/HinfI restriction enzymes overnight before starting TCA assay and then Southern Blot as described with minor modifications to Phi29 DNA polymerization (MBI Fermentas) with a mammalian telomeric primer and a mammalian telomeric probe for hybridization. .. Blot images were captured and quantified with Storm 840 scanner and ImageQuant TL software (Amersham Biosciences).

    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: 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 ( ).

    Plasmid Preparation:

    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.

    Hybridization:

    Article Title: A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome
    Article Snippet: .. DNA was double digested by AluI/HinfI restriction enzymes overnight before starting TCA assay and then Southern Blot as described with minor modifications to Phi29 DNA polymerization (MBI Fermentas) with a mammalian telomeric primer and a mammalian telomeric probe for hybridization. .. Blot images were captured and quantified with Storm 840 scanner and ImageQuant TL software (Amersham Biosciences).

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  • 99
    Thermo Fisher phi29 reaction buffer
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Reaction Buffer, 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/phi29 reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phi29 reaction buffer - by Bioz Stars, 2020-11
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    98
    Thermo Fisher transcriptase reaction
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Transcriptase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transcriptase reaction/product/Thermo Fisher
    Average 98 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    transcriptase reaction - by Bioz Stars, 2020-11
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    Image Search Results


    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

    Techniques: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

    Techniques: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

    Techniques: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated