phi29 dna polymerase reaction buffer  (Thermo Fisher)


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    Name:
    Reaction Buffer
    Description:
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    Catalog Number:
    b43
    Price:
    None
    Applications:
    In Vitro Transcription|One-Step qRT-PCR|PCR & Real-Time PCR|RT-PCR|Real Time PCR (qPCR)|Reverse Transcription|Two-Step RT-PCR|Gene Expression Analysis & Genotyping
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    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher phi29 dna polymerase reaction buffer
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    https://www.bioz.com/result/phi29 dna polymerase reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase reaction buffer - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Conjugation Assay:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Amplification:

    Article Title: Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications
    Article Snippet: .. ChIP-derived DNA was amplified in a 30 µL PCR reaction containing 1x DreamTaq Buffer (Fermentas GmbH, Leon-Rot, Germany), 200 µM of each dNTP (Takara, Shiga, Japan), 1% of DMSO (Merck Millipore, Darmstadt, Germany), 5 µM of each primer, and 25 units of DreamTaq Polymerase (Fermentas). .. MAGEA1 amplification was performed for 36 cycles : 45 sec at 94°C, 45 sec at 66°C, 70 sec at 72°C.

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

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    Polymerase Chain Reaction:

    Article Title: Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications
    Article Snippet: .. ChIP-derived DNA was amplified in a 30 µL PCR reaction containing 1x DreamTaq Buffer (Fermentas GmbH, Leon-Rot, Germany), 200 µM of each dNTP (Takara, Shiga, Japan), 1% of DMSO (Merck Millipore, Darmstadt, Germany), 5 µM of each primer, and 25 units of DreamTaq Polymerase (Fermentas). .. MAGEA1 amplification was performed for 36 cycles : 45 sec at 94°C, 45 sec at 66°C, 70 sec at 72°C.

    Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
    Article Snippet: .. For RT–PCR, ∼5 μg of total RNA were treated with 5 units of RNase-free DNase (Boehringer-Mannheim) in 1× PCR buffer (GIBCO-BRL) containing 2.5 m m MgCl2 at 37°C for 30 min. After heat inactivation at 80°C for 5 min, samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with ethanol. .. The RNA was reverse transcribed using 5 pmoles of random hexamers (Pharmacia Biotech) in a 12-μl reaction containing 1× PCR buffer (GIBCO-BRL), 2.1 m m MgCl2 , 0.5 m m of each deoxynucleotide triphosphate (dNTP), 10 m m dithiothreitol, and 120 units of Superscript II reverse transcriptase (GIBCO-BRL) by incubating at 25°C for 10 min followed by 42°C for 55 min and heat inactivation at 70°C for 20 min.

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    SYBR Green Assay:

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Concentration Assay:

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

    Incubation:

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

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

    Article Title: Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats
    Article Snippet: .. Briefly, unfixed cryosections were incubated for 15 min in calpain reaction buffer (CRB; 25 mM HEPES, 65 mM KCl, 2 mM MgCl2 , 1,5 mM CaCl2 , 2 mM DTT) and then sections were incubated at 35°C for 1 h in the dark in 2 µM fluorescent calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl- L-methionine amide (CMAC, t-BOC-Leu-Met; Molecular Probes, Inc. Eugene, USA). .. Fluorescence was generated by calpain-dependent cleavage of t-Boc-Leu-Met-CMAC.

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
    Article Snippet: .. For RT–PCR, ∼5 μg of total RNA were treated with 5 units of RNase-free DNase (Boehringer-Mannheim) in 1× PCR buffer (GIBCO-BRL) containing 2.5 m m MgCl2 at 37°C for 30 min. After heat inactivation at 80°C for 5 min, samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with ethanol. .. The RNA was reverse transcribed using 5 pmoles of random hexamers (Pharmacia Biotech) in a 12-μl reaction containing 1× PCR buffer (GIBCO-BRL), 2.1 m m MgCl2 , 0.5 m m of each deoxynucleotide triphosphate (dNTP), 10 m m dithiothreitol, and 120 units of Superscript II reverse transcriptase (GIBCO-BRL) by incubating at 25°C for 10 min followed by 42°C for 55 min and heat inactivation at 70°C for 20 min.

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

    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.

    Chromatin Immunoprecipitation:

    Article Title: Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications
    Article Snippet: .. ChIP-derived DNA was amplified in a 30 µL PCR reaction containing 1x DreamTaq Buffer (Fermentas GmbH, Leon-Rot, Germany), 200 µM of each dNTP (Takara, Shiga, Japan), 1% of DMSO (Merck Millipore, Darmstadt, Germany), 5 µM of each primer, and 25 units of DreamTaq Polymerase (Fermentas). .. MAGEA1 amplification was performed for 36 cycles : 45 sec at 94°C, 45 sec at 66°C, 70 sec at 72°C.

    Plasmid Preparation:

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

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  • 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, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher reaction buffer for phi29 dna polymerase 10x
    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.
    Reaction Buffer For Phi29 Dna Polymerase 10x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reaction buffer for phi29 dna polymerase 10x/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    reaction buffer for phi29 dna polymerase 10x - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    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

    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