phi29 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
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher phi29 reaction buffer
    RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that <t>phi29</t> DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.
    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 reaction buffer/product/Thermo Fisher
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    phi29 reaction buffer - by Bioz Stars, 2020-11
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    Images

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku737

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

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

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

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

    Model of template switching and its implications on ds/ssDNA production. ( A ), ( B ) The template switching event in detail. (A) According to the current view on RCA mechanisms, a polymerase, in grey, produces new DNA (in green) from the circular template (in blue) and continually displaces the 5′-end DNA from the template (in red). We propose that strand switching events, that are randomly distributed with an exponential distribution, converts some of the normal RCA templates into templates for double-stranded DNA production in a mechanism like the one depicted in (B)—switching from the circular template, up to the displaced strand. ( C ) This mechanism can be shut down by single-stranded DNA binding proteins (SSBs) that prevent access to the displaced strand for the polymerase. ( D ) Phases of ssDNA versus dsDNA production of a single template. Initially, all templates are assumed to produce ssDNA in our model. Assuming that a template switching event occurs at time t 0 /2, then the polymerase attached to this template will consume the ssDNA that has been produced up to that time while converting it to dsDNA. Assuming that the rate of the polymerase is equal while processing along the single strand, at time t 0 it will stop nucleotide incorporation and fall of the template that is now fully double-stranded. ( E ) Assuming an exponential decay of the templates into dsDNA production with a rate constant of λ , the probability density function will determine the instantaneous flipping probability. To get a figure of the current production rate of dsDNA at time t , one needs to consider that only the templates that have switched between t /2 and t will be currently making dsDNA. The rate of ds-production at t is thus proportional to an integral over the probability density function between t /2 and t times the concentration, n , and the phi29 rate, ø . Once the rate of dsDNA production is known, the rate of ssDNA production can be calculated as the rate of production of all non-switched templates, minus the above ds-DNA rate (because that process consumes already produced ssDNA). Integrating (Supplementary Note S1) the rates, yields expressions for the total mass of produced dsDNA, Nds ( t ), and total mass of produced ssDNA, Nss ( t ). The expressions are plotted in ( F ) using arbitrary units. ( G ) Quantitative fit of the data in Figure 3C to the theoretical curves ( Nds ( t ) and Nss ( t ) not fitted independently with respect to each other) reveals a close resemblance to the measured data and our model. From this we estimate the rate constant λ to be 1.95 ± 0.25 × 10 −5 events per second per template for the switching process.
    Figure Legend Snippet: Model of template switching and its implications on ds/ssDNA production. ( A ), ( B ) The template switching event in detail. (A) According to the current view on RCA mechanisms, a polymerase, in grey, produces new DNA (in green) from the circular template (in blue) and continually displaces the 5′-end DNA from the template (in red). We propose that strand switching events, that are randomly distributed with an exponential distribution, converts some of the normal RCA templates into templates for double-stranded DNA production in a mechanism like the one depicted in (B)—switching from the circular template, up to the displaced strand. ( C ) This mechanism can be shut down by single-stranded DNA binding proteins (SSBs) that prevent access to the displaced strand for the polymerase. ( D ) Phases of ssDNA versus dsDNA production of a single template. Initially, all templates are assumed to produce ssDNA in our model. Assuming that a template switching event occurs at time t 0 /2, then the polymerase attached to this template will consume the ssDNA that has been produced up to that time while converting it to dsDNA. Assuming that the rate of the polymerase is equal while processing along the single strand, at time t 0 it will stop nucleotide incorporation and fall of the template that is now fully double-stranded. ( E ) Assuming an exponential decay of the templates into dsDNA production with a rate constant of λ , the probability density function will determine the instantaneous flipping probability. To get a figure of the current production rate of dsDNA at time t , one needs to consider that only the templates that have switched between t /2 and t will be currently making dsDNA. The rate of ds-production at t is thus proportional to an integral over the probability density function between t /2 and t times the concentration, n , and the phi29 rate, ø . Once the rate of dsDNA production is known, the rate of ssDNA production can be calculated as the rate of production of all non-switched templates, minus the above ds-DNA rate (because that process consumes already produced ssDNA). Integrating (Supplementary Note S1) the rates, yields expressions for the total mass of produced dsDNA, Nds ( t ), and total mass of produced ssDNA, Nss ( t ). The expressions are plotted in ( F ) using arbitrary units. ( G ) Quantitative fit of the data in Figure 3C to the theoretical curves ( Nds ( t ) and Nss ( t ) not fitted independently with respect to each other) reveals a close resemblance to the measured data and our model. From this we estimate the rate constant λ to be 1.95 ± 0.25 × 10 −5 events per second per template for the switching process.

    Techniques Used: DNA Binding Assay, Produced, Concentration Assay

    2) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    3) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    4) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

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    Conjugation Assay:

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    Amplification:

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    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
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    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
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    Purification:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
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    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).

    Incubation:

    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: 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.

    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: 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.

    Staining:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    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.

    SDS Page:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Molecular Weight:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

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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
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    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
    98/100 stars
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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

    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

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Journal: Micromachines

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    doi: 10.3390/mi8020062

    Figure Lengend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    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).

    Techniques: Multiple Displacement Amplification, Transmission Electron Microscopy