Structured Review

Thermo Fisher dntp
Effects of the next complementary <t>dNTP</t> on DNase I protection and stable complex formation by <t>HIV-1</t> RT
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Images

1) Product Images from "Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet"

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

Journal:

doi: 10.1016/j.jmb.2007.03.006

Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT
Figure Legend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

Techniques Used:

2) Product Images from "Efficient in situ barcode sequencing using padlock probe-based BaristaSeq"

Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1206

Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).
Figure Legend Snippet: Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

Techniques Used: In Situ, Sequencing, Amplification, Infection, In Vitro

3) Product Images from "Direct Cell Lysis for Single-Cell Gene Expression Profiling"

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2013.00274

Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Polymerase Chain Reaction

4) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-21

Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

5) Product Images from "Mechanical properties of DNA-like polymers"

Article Title: Mechanical properties of DNA-like polymers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt808

Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

6) Product Images from "Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline"

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

Journal: AoB Plants

doi: 10.1093/aobpla/pls033

Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

Techniques Used: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

Techniques Used: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

7) Product Images from "Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *"

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.009506

The F-helix exhibits reduced HDX rates during ternary complex formation that are independent of dNTP identity. A , the kinetic profile for HDX in residues 151–170 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP
Figure Legend Snippet: The F-helix exhibits reduced HDX rates during ternary complex formation that are independent of dNTP identity. A , the kinetic profile for HDX in residues 151–170 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP

Techniques Used:

Changes in HDX kinetics for the thumb domain are sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 180–202 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles
Figure Legend Snippet: Changes in HDX kinetics for the thumb domain are sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 180–202 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles

Techniques Used:

The kinetics of HDX in the finger domain is sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 48–63 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles ), +DNA/Mg
Figure Legend Snippet: The kinetics of HDX in the finger domain is sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 48–63 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles ), +DNA/Mg

Techniques Used:

Overview of Changes in HDX as a Function of Primer/Template DNA, MgCl2 , and Incoming dNTP
Figure Legend Snippet: Overview of Changes in HDX as a Function of Primer/Template DNA, MgCl2 , and Incoming dNTP

Techniques Used:

8) Product Images from "Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa"

Article Title: Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa

Journal: The FEBS journal

doi: 10.1111/febs.13304

Proposed reaction pathway for DinB and pol κ. The free pol binds DNA which results in a relatively closed and stable binary complex. In the case of correct dNTP incorporation opposite preferred DNA bases, there is a conserved conformational change
Figure Legend Snippet: Proposed reaction pathway for DinB and pol κ. The free pol binds DNA which results in a relatively closed and stable binary complex. In the case of correct dNTP incorporation opposite preferred DNA bases, there is a conserved conformational change

Techniques Used:

DinB and pol κ are protected from exchange in the presence of undamaged DNA and the correct dNTP. (a) Minimal model for nucleotide incorporation. The free pol is denoted E, the binary pol-DNA complex ED, and the ternary pol-DNA-dNTP complex EDN.
Figure Legend Snippet: DinB and pol κ are protected from exchange in the presence of undamaged DNA and the correct dNTP. (a) Minimal model for nucleotide incorporation. The free pol is denoted E, the binary pol-DNA complex ED, and the ternary pol-DNA-dNTP complex EDN.

Techniques Used:

9) Product Images from "Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions"

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions

Journal: The American Journal of Pathology

doi:

AmpliSeq. A: Theoretical AmpliSeq reaction. Because of supplemental dNTPs, PCR amplification is supported during initial cycles of the reaction. As PCR occurs, dNTPs are consumed resulting in an increase in the ddNTP/dNTP ratio. This causes the reaction to convert itself to cycle sequencing in latter cycles. B: Anticipated PCR product generated during AmpliSeq. Forward and reverse primer sequences are shown in dark blue and the rest of the PCR product in light blue. In the reverse primer, dots denote the abasic region and stripes , the nontemplated thymidines. Yellow highlight designates the mutation site ( asterisk ). C: Bidirectional AmpliSeq results of a factor V wild-type homozygote. D: Unidirectional AmpliSeq of a factor V wild-type homozygote. Yellow highlighting indicates the potential mutation site in sequencing products.
Figure Legend Snippet: AmpliSeq. A: Theoretical AmpliSeq reaction. Because of supplemental dNTPs, PCR amplification is supported during initial cycles of the reaction. As PCR occurs, dNTPs are consumed resulting in an increase in the ddNTP/dNTP ratio. This causes the reaction to convert itself to cycle sequencing in latter cycles. B: Anticipated PCR product generated during AmpliSeq. Forward and reverse primer sequences are shown in dark blue and the rest of the PCR product in light blue. In the reverse primer, dots denote the abasic region and stripes , the nontemplated thymidines. Yellow highlight designates the mutation site ( asterisk ). C: Bidirectional AmpliSeq results of a factor V wild-type homozygote. D: Unidirectional AmpliSeq of a factor V wild-type homozygote. Yellow highlighting indicates the potential mutation site in sequencing products.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Generated, Mutagenesis

Related Articles

Clone Assay:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France).

Amplification:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: Paragraph title: PCR amplification and sequence analysis ... All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler. .. All reactions were performed in duplicates in a total reaction volume of 10 μL containing SYBR Green I Master mix (Roche, Amsterdam, Holland), ddH2 O and 5 pmol/μL of each target forward and reverse primer, under the following conditions: pre-incubation at 95° for 10 min followed by cycled amplification at 95° for 10 s, annealing for 20 s, and 72° for 5 s for 50 cycles.

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: Paragraph title: Polymerase chain reaction amplification of modified DNAs ... For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: PCR amplification of each of the 4 single stranded DNA sequences by using primers consisting of the stabilization and the restriction site sequences (italics in ). .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France).

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: .. PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl. .. Primers for 16S rRNA gene amplification were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′).

Mass Spectrometry:

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *
Article Snippet: All oligonucleotides used in this work were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified by the manufacturer using high-performance liquid chromatography, with analysis by matrix-assisted laser desorption time-of-flight MS. .. All of the MgCl2 , dNTP, and DNA stock solutions used in the HDX analysis were lyophilized to dryness and resuspended in D2 O (100%, Acros Organics, Geel, Belgium).

Synthesized:

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
Article Snippet: PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. .. All oligonucleotides were synthesized and purified by Oligo’s Etc. (Wilsonville, OR).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France).

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *
Article Snippet: All oligonucleotides used in this work were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified by the manufacturer using high-performance liquid chromatography, with analysis by matrix-assisted laser desorption time-of-flight MS. .. All of the MgCl2 , dNTP, and DNA stock solutions used in the HDX analysis were lyophilized to dryness and resuspended in D2 O (100%, Acros Organics, Geel, Belgium).

Construct:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Three plasmids were constructed such as CatA for category A agents, CatB for category B agents and CatVEF for vesicular eruptive fever (Tables and ). .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France).

Real-time Polymerase Chain Reaction:

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: Paragraph title: RNA extraction and gene expression analysis by real-time quantitative PCR ... 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler.

Incubation:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
Article Snippet: .. For upstream mapping of the borders of the stable complexes, 5 nM 3'-[32 P] ddAMP-terminated, 5'-phosphorylated L32 primer annealed to excess WL50 template was incubated with 200 nM HIV-1 RT and dNTP, foscarnet or no ligand as indicated in the figures, in 10 μl RB buffer for 15 min at 37°C, followed by incubation at 4°C for 5 min. After addition of 1.2 U lambda exonuclease (USB Corp.), the samples were incubated at 37°C for 8 min followed by heat-inactivation at 90°C for 5 min. For downstream mapping of the borders of the stable complexes, 5 nM 3'-[32 P]ddAMP-labeled WL65 template annealed to excess ddAMP-terminated L32 primer was incubated with 12.5 nM WT HIV-1 RT and ligand (0.8 mM dNTP, 3.2 mM foscarnet, or no ligand) in 10 μl RB buffer for 15 min at 37°C, followed by incubation at 4°C for 5 min. .. Ten units of RecJf (New England Biolabs) were added and the samples were incubated at 37°C for 10 min followed by heat-inactivation at 90°C for 5 min. For both upstream and downstream mapping, 12 μl loading buffer were added and the products were separated by electrophoresis through a denaturing 20% polyacrylamide gel.

Article Title: Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa
Article Snippet: .. Samples containing 3–10 µM protein in the presence or absence of primer/template DNA (2-fold molar excess) and dNTP (1 mM) in 15 µL assay buffer (25 mM Hepes, pH 7.5, 20 mM NaCl, 5 mM MgSO4 , and 3 mM dithiothreitol) were incubated for 30 min at room temperature before addition of 0.5 µL of Sypro Orange (Invitrogen, Grand Island, NY, USA) to a final concentration of 10×. .. Samples were analyzed using a CFX 96 Real-Time system (Bio-Rad, Hercules, CA, USA) as described [ ].

Expressing:

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: Paragraph title: RNA extraction and gene expression analysis by real-time quantitative PCR ... 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler.

Modification:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: Paragraph title: Polymerase chain reaction amplification of modified DNAs ... For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen).

High Performance Liquid Chromatography:

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *
Article Snippet: All oligonucleotides used in this work were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified by the manufacturer using high-performance liquid chromatography, with analysis by matrix-assisted laser desorption time-of-flight MS. .. All of the MgCl2 , dNTP, and DNA stock solutions used in the HDX analysis were lyophilized to dryness and resuspended in D2 O (100%, Acros Organics, Geel, Belgium).

Polymerase Chain Reaction:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: .. All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume. .. Polymerase chain reaction primer sequences for LSU rDNA and optimized annealing temperatures are specified in Table .

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: .. 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler. .. All reactions were performed in duplicates in a total reaction volume of 10 μL containing SYBR Green I Master mix (Roche, Amsterdam, Holland), ddH2 O and 5 pmol/μL of each target forward and reverse primer, under the following conditions: pre-incubation at 95° for 10 min followed by cycled amplification at 95° for 10 s, annealing for 20 s, and 72° for 5 s for 50 cycles.

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: .. Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen). ..

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
Article Snippet: .. PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. ..

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: .. For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: .. PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl. .. Primers for 16S rRNA gene amplification were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′).

Imaging:

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen). .. Images of bands were acquired by the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantification was performed using the built-in Image Lab software.

Sequencing:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: Paragraph title: PCR amplification and sequence analysis ... All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *
Article Snippet: The template DNA sequence was 5′-TCACGGAATCCTTCCCCC-3′. .. All of the MgCl2 , dNTP, and DNA stock solutions used in the HDX analysis were lyophilized to dryness and resuspended in D2 O (100%, Acros Organics, Geel, Belgium).

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: Paragraph title: Identification of the isolate by 16S rRNA sequence analysis ... PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl.

Recombinant:

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline
Article Snippet: .. All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume. .. Polymerase chain reaction primer sequences for LSU rDNA and optimized annealing temperatures are specified in Table .

Mutagenesis:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: Polymerase chain reaction amplification of modified DNAs pUC19-based plasmids containing intrinsically straight ∼200-bp sequences ( ) were subjected to site-directed mutagenesis to obtain suitable restriction sites ( Supplementary Data S2 ). .. For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen).

Isolation:

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: Semiquantitative reverse transcription-PCR (RT-PCR) Total RNAs were isolated from A375 cells via the TRIzol method following the vendor’s manual (Invitrogen; Thermo Fisher Scientific) and quantified by NanoDrop. .. Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen).

Size-exclusion Chromatography:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Purification:

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
Article Snippet: PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. .. The PCR products were identified on 10% polyacrylamide gel electrophoresis and then purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: .. For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *
Article Snippet: All oligonucleotides used in this work were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified by the manufacturer using high-performance liquid chromatography, with analysis by matrix-assisted laser desorption time-of-flight MS. .. All of the MgCl2 , dNTP, and DNA stock solutions used in the HDX analysis were lyophilized to dryness and resuspended in D2 O (100%, Acros Organics, Geel, Belgium).

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: Identification of the isolate by 16S rRNA sequence analysis DNAeasy Tissue kit (Qiagen, Valencia, CA, USA) was used to extract DNA from the cultures of purified isolates, according to the manufacturer's instructions. .. PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: Paragraph title: Semiquantitative reverse transcription-PCR (RT-PCR) ... Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen).

Polyacrylamide Gel Electrophoresis:

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
Article Snippet: PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. .. The PCR products were identified on 10% polyacrylamide gel electrophoresis and then purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

Lysis:

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling
Article Snippet: .. The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco). ..

Plasmid Preparation:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: .. For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France).

Software:

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler. .. Target genes were normalised to expression levels of stabile housekeeping gene GAPDH, calculated by Normfinder software [ ]. mRNA ratios of target gene/house-keeping gene were normalized to untreated control. contains forward/reverse primers and primer specific annealing temperatures ( ).

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen). .. Images of bands were acquired by the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantification was performed using the built-in Image Lab software.

SYBR Green Assay:

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler. .. All reactions were performed in duplicates in a total reaction volume of 10 μL containing SYBR Green I Master mix (Roche, Amsterdam, Holland), ddH2 O and 5 pmol/μL of each target forward and reverse primer, under the following conditions: pre-incubation at 95° for 10 min followed by cycled amplification at 95° for 10 s, annealing for 20 s, and 72° for 5 s for 50 cycles.

RNA Extraction:

Article Title: 25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages
Article Snippet: Paragraph title: RNA extraction and gene expression analysis by real-time quantitative PCR ... 100 ng of total RNA and in total of 40 μL reaction mixtures of 1x PCR buffer, 6.25 mM MgCL2 , 2.5 μM Oligo(dT), 1 mM dNTP, 2.5 units/μL RT, 1 unit/μL RNase inhibitor (ThermoFisher, Hvidovre, Denmark) and ddH2 O were synthesised to cDNA by GeneAmp PCR System 9600 thermal cycler.

Agarose Gel Electrophoresis:

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl. .. The PCR products were confirmed by electrophoresis on an agarose gel and visualized under UV light after SYBRSafe (Invitrogen) staining.

In Vitro:

Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq
Article Snippet: .. In vitro gap-filling assay In vitro gap-filling assays were performed in 1X Ampligase buffer (Epicentre) with 10 nM padlock probes (XC1149 and XC1151) and 10 nM cDNA template (XC1498), 20 μM dNTP, 0.012 U/μl Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) or Stoffel fragment (DNA Gdansk), additional 50 mM KCl, 20% formamide, and glycerol to a final concentration of 10%. .. The reaction was kept at 37°C for 30 min. We then terminated the reactions by adding 2× TBE Urea sample buffer (Thermo Fisher Scientific) and ran the samples on 15% Novex TBE–Urea gels (Thermo Fisher Scientific).

Electrophoresis:

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
Article Snippet: For upstream mapping of the borders of the stable complexes, 5 nM 3'-[32 P] ddAMP-terminated, 5'-phosphorylated L32 primer annealed to excess WL50 template was incubated with 200 nM HIV-1 RT and dNTP, foscarnet or no ligand as indicated in the figures, in 10 μl RB buffer for 15 min at 37°C, followed by incubation at 4°C for 5 min. After addition of 1.2 U lambda exonuclease (USB Corp.), the samples were incubated at 37°C for 8 min followed by heat-inactivation at 90°C for 5 min. For downstream mapping of the borders of the stable complexes, 5 nM 3'-[32 P]ddAMP-labeled WL65 template annealed to excess ddAMP-terminated L32 primer was incubated with 12.5 nM WT HIV-1 RT and ligand (0.8 mM dNTP, 3.2 mM foscarnet, or no ligand) in 10 μl RB buffer for 15 min at 37°C, followed by incubation at 4°C for 5 min. .. Ten units of RecJf (New England Biolabs) were added and the samples were incubated at 37°C for 10 min followed by heat-inactivation at 90°C for 5 min. For both upstream and downstream mapping, 12 μl loading buffer were added and the products were separated by electrophoresis through a denaturing 20% polyacrylamide gel.

Article Title: Photothermal effects of NaYF4:Yb,Er@PE3@Fe3O4 superparamagnetic nanoprobes in the treatment of melanoma
Article Snippet: Benchtop PCR was performed on a GeneAmp PCR System 9700 machine in a 50 µl reaction volume: 1× PCR buffer (+Mg2+ ), 0.2 mM dNTP, 0.2 µM primers, 50 ng of template cDNA, and 0.5 µL of Platinum TaqDNA polymerase (Invitrogen). .. The PCR products were resolved by electrophoresis using agarose gels supplemented with ethidium bromide.

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl. .. The PCR products were confirmed by electrophoresis on an agarose gel and visualized under UV light after SYBRSafe (Invitrogen) staining.

Concentration Assay:

Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq
Article Snippet: .. In vitro gap-filling assay In vitro gap-filling assays were performed in 1X Ampligase buffer (Epicentre) with 10 nM padlock probes (XC1149 and XC1151) and 10 nM cDNA template (XC1498), 20 μM dNTP, 0.012 U/μl Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) or Stoffel fragment (DNA Gdansk), additional 50 mM KCl, 20% formamide, and glycerol to a final concentration of 10%. .. The reaction was kept at 37°C for 30 min. We then terminated the reactions by adding 2× TBE Urea sample buffer (Thermo Fisher Scientific) and ran the samples on 15% Novex TBE–Urea gels (Thermo Fisher Scientific).

Article Title: Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa
Article Snippet: .. Samples containing 3–10 µM protein in the presence or absence of primer/template DNA (2-fold molar excess) and dNTP (1 mM) in 15 µL assay buffer (25 mM Hepes, pH 7.5, 20 mM NaCl, 5 mM MgSO4 , and 3 mM dithiothreitol) were incubated for 30 min at room temperature before addition of 0.5 µL of Sypro Orange (Invitrogen, Grand Island, NY, USA) to a final concentration of 10×. .. Samples were analyzed using a CFX 96 Real-Time system (Bio-Rad, Hercules, CA, USA) as described [ ].

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
Article Snippet: .. PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. ..

Staining:

Article Title: The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus
Article Snippet: PCR amplification was performed in a thermocycler (Gene Technologies, Braintree, United Kingdom) in a total volume of 50 μl, containing 1.0 μl (40 ng) of DNA, 10 μl of 5X Phire reaction buffer, 200 μM of each dNTP (1.0 μl), 10 μM of each primer (2.0 μl), 0.5 μl of Phire Hot Start II polymerase (Thermo Scientific, USA), and nuclease-free water (Promega) up to 50 μl. .. The PCR products were confirmed by electrophoresis on an agarose gel and visualized under UV light after SYBRSafe (Invitrogen) staining.

Variant Assay:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

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  • 99
    Thermo Fisher dntp
    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ <t>cDNA)</t> or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated <t>dNTP</t> concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher fluorescein labelled dntp s
    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of <t>dNTP’s</t> on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .
    Fluorescein Labelled Dntp S, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Journal: Nucleic Acids Research

    Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

    doi: 10.1093/nar/gkx1206

    Figure Lengend Snippet: Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Article Snippet: In vitro gap-filling assay In vitro gap-filling assays were performed in 1X Ampligase buffer (Epicentre) with 10 nM padlock probes (XC1149 and XC1151) and 10 nM cDNA template (XC1498), 20 μM dNTP, 0.012 U/μl Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) or Stoffel fragment (DNA Gdansk), additional 50 mM KCl, 20% formamide, and glycerol to a final concentration of 10%.

    Techniques: In Situ, Sequencing, Amplification, Infection, In Vitro

    Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Journal: Scientific Reports

    Article Title: Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

    doi: 10.1038/srep39985

    Figure Lengend Snippet: Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Article Snippet: dNTP Quantification by UPLC-MS/MS Chromatography and Mass Spectrometry Method Analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic AX 5 μm, 100 × 2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a mixture of 10 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 6.0 (buffer A), and 1 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 10.5 (buffer B).

    Techniques: Activity Assay, In Vitro, Incubation, Colorimetric Assay, Expressing, Mass Spectrometry

    Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: Flow Cytometry, Cytometry

    ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: High Performance Liquid Chromatography, Labeling

    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Infection, Binding Assay, Fluorescence

    The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Fluorescence, Binding Assay

    ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Recombinant