phi29 polymerase buffer  (Thermo Fisher)


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  • 98
    Name:
    Reaction Buffer for phi29 DNA Polymerase 10X
    Description:
    Thermo Scientific 10X Buffer for phi29 DNA Polymerase is the optimal buffer recommended for use with highly processive phi 29 DNA polymerase
    Catalog Number:
    b62
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis|Gene Synthesis, Seamless Cloning, & Assembly|Seamless Cloning & Genetic Assembly
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher phi29 polymerase buffer
    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, <t>phi29</t> DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Thermo Scientific 10X Buffer for phi29 DNA Polymerase is the optimal buffer recommended for use with highly processive phi 29 DNA polymerase
    https://www.bioz.com/result/phi29 polymerase buffer/product/Thermo Fisher
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phi29 polymerase buffer - by Bioz Stars, 2020-11
    98/100 stars

    Images

    1) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    2) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    Related Articles

    Amplification:

    Article Title: Ictal onset sites and GABAergic neuron loss in epileptic pilocarpine-treated rats
    Article Snippet: .. Amplification solution contained 1000 units/ml NxGen phi29 DNA polymerase in phi29 polymerase buffer (Lucigen) and 1 mM dNTP mix (0.25 mM/nucleotide, Invitrogen), 0.4 units/μl RNaseOUT, 5% glycerol, and 0.4 mg/ml BSA; sections incubated overnight at 37°C. .. The next day sections were rinsed in a label probe hybridization buffer of 2x saline-sodium citrate (Invitrogen) and 20% deionized formamide (Invitrogen) in DEPC-treated water.

    Article Title: Ictal onset sites and GABAergic neuron loss in epileptic pilocarpine-treated rats
    Article Snippet: .. Sections were rinsed in hybridization buffer followed by phi29 polymerase buffer before the rolling circle amplification reaction. .. Amplification solution contained 1000 units/ml NxGen phi29 DNA polymerase in phi29 polymerase buffer (Lucigen) and 1 mM dNTP mix (0.25 mM/nucleotide, Invitrogen), 0.4 units/μl RNaseOUT, 5% glycerol, and 0.4 mg/ml BSA; sections incubated overnight at 37°C.

    Cell Culture:

    Article Title: Immune-privileged embryonic Swiss mouse STO and STO cell-derived progenitor cells: major histocompatibility complex and cell differentiation antigen expression patterns resemble those of human embryonic stem cell lines
    Article Snippet: .. STO and SCD lines 3(8)21 and 3(8)21-EGFP were cultured by standard procedures; , passage numbers were recorded and cell counts performed as described elsewhere., Swiss mouse q-haplotype NIH3T3 cells, d-haplotype BALB/c 3T3 cells, human embryonic kidney 293 cells (American Type Culture Collection #CRL-1573), mouse H-2Kb/d -, H-2Db/d - and H-2Lb/d -positive B6-2 cells and primary BALB/c kidney cells prepared from trypsinized tissue were cultured as described previously., Fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) was heat-inactivated at 56° for 30 min. .. Unless noted, cells were cultured in 3- or 10-cm-diameter plastic tissue culture dishes (BD Falcon™, Bedford, MA).

    TaqMan microRNA Assay:

    Article Title: Human hepatocellular carcinoma cell-specific miRNAs reveal the differential expression of miR-24 and miR-27a in cirrhotic/non-cirrhotic HCC
    Article Snippet: .. The reverse transcriptase reaction was performed by incubating the samples at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Each PCR reaction (20 μ l) contained 1.3 μ l of reverse transcriptase product, 10 μ l of Taq-Man 2X Universal PCR Master Mix, and 1 μ l of the appropriate TaqMan MicroRNA Assay solution containing primers and probes for each miR of interest. .. The PCR mixtures were incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. PCRs were performed in triplicate using a 7500 Real-Time PCR system.

    Concentration Assay:

    Article Title: Recording the age of RNA with deamination
    Article Snippet: .. Cell concentration was counted and the appropriate volume of cells was added to a 10x reaction for a targeted cell recovery of 2000 cells. .. The reaction was run through a 10x chip according to the 10x Genomics Chromium Single Cell protocol.

    Incubation:

    Article Title: Ictal onset sites and GABAergic neuron loss in epileptic pilocarpine-treated rats
    Article Snippet: .. Amplification solution contained 1000 units/ml NxGen phi29 DNA polymerase in phi29 polymerase buffer (Lucigen) and 1 mM dNTP mix (0.25 mM/nucleotide, Invitrogen), 0.4 units/μl RNaseOUT, 5% glycerol, and 0.4 mg/ml BSA; sections incubated overnight at 37°C. .. The next day sections were rinsed in a label probe hybridization buffer of 2x saline-sodium citrate (Invitrogen) and 20% deionized formamide (Invitrogen) in DEPC-treated water.

    other:

    Article Title: microRNA-944 overexpression is a biomarker for poor prognosis of advanced cervical cancer
    Article Snippet: The reverse transcriptase reaction was carried out at 25 °C for 10 min, at 37 °C for 50 min, and at 70 °C for 15 min.

    Article Title: MicroRNA-103-1 Selectively Downregulates Brain NCX1 and Its Inhibition by Anti-miRNA Ameliorates Stroke Damage and Neurological Deficits
    Article Snippet: The semiquantitative polymerase reaction was performed as previously described.

    Polymerase Chain Reaction:

    Article Title: Human hepatocellular carcinoma cell-specific miRNAs reveal the differential expression of miR-24 and miR-27a in cirrhotic/non-cirrhotic HCC
    Article Snippet: .. The reverse transcriptase reaction was performed by incubating the samples at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Each PCR reaction (20 μ l) contained 1.3 μ l of reverse transcriptase product, 10 μ l of Taq-Man 2X Universal PCR Master Mix, and 1 μ l of the appropriate TaqMan MicroRNA Assay solution containing primers and probes for each miR of interest. .. The PCR mixtures were incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. PCRs were performed in triplicate using a 7500 Real-Time PCR system.

    Hybridization:

    Article Title: Ictal onset sites and GABAergic neuron loss in epileptic pilocarpine-treated rats
    Article Snippet: .. Sections were rinsed in hybridization buffer followed by phi29 polymerase buffer before the rolling circle amplification reaction. .. Amplification solution contained 1000 units/ml NxGen phi29 DNA polymerase in phi29 polymerase buffer (Lucigen) and 1 mM dNTP mix (0.25 mM/nucleotide, Invitrogen), 0.4 units/μl RNaseOUT, 5% glycerol, and 0.4 mg/ml BSA; sections incubated overnight at 37°C.

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  • 98
    Thermo Fisher phi29 polymerase buffer
    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, <t>phi29</t> DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Phi29 Polymerase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 polymerase buffer/product/Thermo Fisher
    Average 98 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    phi29 polymerase buffer - by Bioz Stars, 2020-11
    98/100 stars
      Buy from Supplier

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

    Image Search Results


    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Journal: Scientific Reports

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    doi: 10.1038/s41598-018-23582-1

    Figure Lengend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Article Snippet: The slides were then washed twice in TBS-T for 3 min. Circularization oligonucleotides for the two probe designs were ligated using 0.02 U/µl T4 DNA ligase (Thermo Scientific) in T4 DNA ligase buffer supplemented with 0.25 mg/ml BSA for 30 min at 37 °C, thereafter the slides were washed twice for 3 min in TBS-T. RCA was performed by addition of 0.5 U/µl phi29 polymerase (Thermo scientific) in phi29 polymerase buffer (Thermo Scientific) supplemented with 7.5 ng/ml PolyA (Sigma-Aldrich), 0.25 mM dNTP and 0.25 mg/ml BSA for 60 min at 37 °C, followed by two 3 min washes in TBS-T.

    Techniques: In Situ, Proximity Ligation Assay, Incubation

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Journal: Scientific Reports

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    doi: 10.1038/s41598-018-23582-1

    Figure Lengend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Article Snippet: The slides were then washed twice in TBS-T for 3 min. Circularization oligonucleotides for the two probe designs were ligated using 0.02 U/µl T4 DNA ligase (Thermo Scientific) in T4 DNA ligase buffer supplemented with 0.25 mg/ml BSA for 30 min at 37 °C, thereafter the slides were washed twice for 3 min in TBS-T. RCA was performed by addition of 0.5 U/µl phi29 polymerase (Thermo scientific) in phi29 polymerase buffer (Thermo Scientific) supplemented with 7.5 ng/ml PolyA (Sigma-Aldrich), 0.25 mM dNTP and 0.25 mg/ml BSA for 60 min at 37 °C, followed by two 3 min washes in TBS-T.

    Techniques: In Situ, Proximity Ligation Assay, Incubation

    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