Structured Review

Roche dntps
Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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Images

1) Product Images from "Molecular dissection of the domain architecture and catalytic activities of human PrimPol"

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku214

Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
Figure Legend Snippet: Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

Techniques Used: Activity Assay, Functional Assay

2) Product Images from "Molecular dissection of the domain architecture and catalytic activities of human PrimPol"

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku214

Template-independent extension in the presence of manganese. Human PrimPol was incubated for 30 min with DNA substrate and each of the dNTPs in the presence of manganese. ( A ) Wild-type PrimPol was unable to extend from a ds DNA template with a blunt end, but could extend from a primer annealed to an overhanging template, even synthesising long tracts of homopolymers. ( B ) PrimPol ZF-KO could extend from an overhanging DNA template in the presence of manganese and, consistent with the wild-type, did not extend from a ds DNA substrate. The incorporation of 1 or 2 nucleotides of guanine or adenine opposite an overhanging template suggests that PrimPol ZF-KO incorporates in a low-fidelity template-dependent manner when incubated with overhanging DNA. ( C ) PrimPol 1–487 exhibited a highly similar terminal transferase activity spectrum to the PrimPol ZF-KO . In the presence of manganese, it incorporated bases opposite an overhang in a low fidelity, template-dependent manner. ( D ) PrimPol 1–354 also exhibited low fidelity extension of a primer annealed to an overhanging template in the presence of manganese.
Figure Legend Snippet: Template-independent extension in the presence of manganese. Human PrimPol was incubated for 30 min with DNA substrate and each of the dNTPs in the presence of manganese. ( A ) Wild-type PrimPol was unable to extend from a ds DNA template with a blunt end, but could extend from a primer annealed to an overhanging template, even synthesising long tracts of homopolymers. ( B ) PrimPol ZF-KO could extend from an overhanging DNA template in the presence of manganese and, consistent with the wild-type, did not extend from a ds DNA substrate. The incorporation of 1 or 2 nucleotides of guanine or adenine opposite an overhanging template suggests that PrimPol ZF-KO incorporates in a low-fidelity template-dependent manner when incubated with overhanging DNA. ( C ) PrimPol 1–487 exhibited a highly similar terminal transferase activity spectrum to the PrimPol ZF-KO . In the presence of manganese, it incorporated bases opposite an overhang in a low fidelity, template-dependent manner. ( D ) PrimPol 1–354 also exhibited low fidelity extension of a primer annealed to an overhanging template in the presence of manganese.

Techniques Used: Incubation, Activity Assay

Polymerase activity and fidelity of human PrimPol. ( A ) Human PrimPol was incubated with dNTPs and substrate at 1, 3, 5 and 30 min time points. PrimPol was proficient at extending an undamaged oligonucleotide template using dNTPs. Human PrimPol did not require an intact zinc finger in order to carry out primer extension, as evidenced by the extension of primers by PrimPol ZF-KO and PrimPol 1–354 . PrimPol 1–487 that lacked the unstructured C-terminus of the protein was also polymerase proficient. PrimPol 1–354 exhibited a higher rate of polymerase activity compared to the other constructs. ( B ) Incorporation of nucleotides opposite two templating cytosine bases. PrimPol was incubated for 5 min with the DNA substrate and each of the dNTPs. All four of these PrimPol constructs inserted two guanine nucleotides opposite two cytosines in Watson–Crick base-pairing manner. PrimPol 1–354 could additionally incorporate a single adenine opposite the first cytosine.
Figure Legend Snippet: Polymerase activity and fidelity of human PrimPol. ( A ) Human PrimPol was incubated with dNTPs and substrate at 1, 3, 5 and 30 min time points. PrimPol was proficient at extending an undamaged oligonucleotide template using dNTPs. Human PrimPol did not require an intact zinc finger in order to carry out primer extension, as evidenced by the extension of primers by PrimPol ZF-KO and PrimPol 1–354 . PrimPol 1–487 that lacked the unstructured C-terminus of the protein was also polymerase proficient. PrimPol 1–354 exhibited a higher rate of polymerase activity compared to the other constructs. ( B ) Incorporation of nucleotides opposite two templating cytosine bases. PrimPol was incubated for 5 min with the DNA substrate and each of the dNTPs. All four of these PrimPol constructs inserted two guanine nucleotides opposite two cytosines in Watson–Crick base-pairing manner. PrimPol 1–354 could additionally incorporate a single adenine opposite the first cytosine.

Techniques Used: Activity Assay, Incubation, Construct

Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
Figure Legend Snippet: Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

Techniques Used: Activity Assay, Functional Assay

Processivity of the polymerase activity of PrimPol. PrimPol was pre-incubated for 30 min at 37°C with an undamaged DNA primer-template substrate to allow PrimPol to bind to the DNA. The reaction was initiated by the addition of dNTPs and an excess of sonicated herring sperm DNA (trap) and time points taken at 15, 30, 60, 120 and 360 s. ( A ) After 360 s, wild-type PrimPol incorporated up to 4 nucleotides opposite the template but a significant fraction of enzyme incorporated only 1, 2 or 3 nucleotides (left panel). To confirm that the trap prevents polymerase extending from a second template, the trap was also added into the pre-incubation mix with PrimPol and the DNA substrate. This reaction was supplemented with dNTPs and there is no extension (right panel), thus successfully exhibiting the effectiveness of the trap. ( B ) PrimPol 1–354 also predominantly incorporates up to 4 nucleotides but there were fewer polymerases incorporating only 1, 2 or 3 nucleotides (left panel). The effectiveness of the trap was also successfully confirmed (right panel). ( C ) Percentage of PrimPol molecules incorporating at least n dNTPs for either full-length PrimPol or PrimPol 1–354 , calculated using Equation (1) .
Figure Legend Snippet: Processivity of the polymerase activity of PrimPol. PrimPol was pre-incubated for 30 min at 37°C with an undamaged DNA primer-template substrate to allow PrimPol to bind to the DNA. The reaction was initiated by the addition of dNTPs and an excess of sonicated herring sperm DNA (trap) and time points taken at 15, 30, 60, 120 and 360 s. ( A ) After 360 s, wild-type PrimPol incorporated up to 4 nucleotides opposite the template but a significant fraction of enzyme incorporated only 1, 2 or 3 nucleotides (left panel). To confirm that the trap prevents polymerase extending from a second template, the trap was also added into the pre-incubation mix with PrimPol and the DNA substrate. This reaction was supplemented with dNTPs and there is no extension (right panel), thus successfully exhibiting the effectiveness of the trap. ( B ) PrimPol 1–354 also predominantly incorporates up to 4 nucleotides but there were fewer polymerases incorporating only 1, 2 or 3 nucleotides (left panel). The effectiveness of the trap was also successfully confirmed (right panel). ( C ) Percentage of PrimPol molecules incorporating at least n dNTPs for either full-length PrimPol or PrimPol 1–354 , calculated using Equation (1) .

Techniques Used: Activity Assay, Incubation, Sonication

PrimPol 1–354 can replicate through CPD and (6–4(PP)) lesions. ( A ) PrimPol 1–354 was incubated with a primer-template substrate in which the template contained a CPD lesion downstream of the primer-template junction in the presence of dNTPs. Time points were taken between 0.5 and 60 min. PrimPol 1–354 extends from the primer up to the CPD, before stalling, it will then added a base opposite the first thymine of the CPD and continue to extend until the end of the template (left panel). PrimPol 1–354 was then incubated with each dNTP to test which nucleotide it incorporates opposite a CPD (right panels). PrimPol 1–354 incorporated adenine opposite the first and second thymine of the CPD. ( B ) The polymerase domain of PrimPol can also perform TLS bypass of a 6–4(PP) lesion immediately downstream of the primer-template junction (left panel). PrimPol 1–354 incorporates either an adenine or cytosine nucleotide opposite the first thymine of the 6–4(PP) (right panels). If an adenine is incorporated opposite the first thymine, a cytosine or thymine is then incorporated opposite the second. If a cytosine is incorporated opposite the first thymine, an adenine, cytosine or thymine will be incorporated opposite the second thymine of the 6–4(PP).
Figure Legend Snippet: PrimPol 1–354 can replicate through CPD and (6–4(PP)) lesions. ( A ) PrimPol 1–354 was incubated with a primer-template substrate in which the template contained a CPD lesion downstream of the primer-template junction in the presence of dNTPs. Time points were taken between 0.5 and 60 min. PrimPol 1–354 extends from the primer up to the CPD, before stalling, it will then added a base opposite the first thymine of the CPD and continue to extend until the end of the template (left panel). PrimPol 1–354 was then incubated with each dNTP to test which nucleotide it incorporates opposite a CPD (right panels). PrimPol 1–354 incorporated adenine opposite the first and second thymine of the CPD. ( B ) The polymerase domain of PrimPol can also perform TLS bypass of a 6–4(PP) lesion immediately downstream of the primer-template junction (left panel). PrimPol 1–354 incorporates either an adenine or cytosine nucleotide opposite the first thymine of the 6–4(PP) (right panels). If an adenine is incorporated opposite the first thymine, a cytosine or thymine is then incorporated opposite the second. If a cytosine is incorporated opposite the first thymine, an adenine, cytosine or thymine will be incorporated opposite the second thymine of the 6–4(PP).

Techniques Used: Incubation

3) Product Images from "PPL2 Translesion Polymerase Is Essential for the Completion of Chromosomal DNA Replication in the African Trypanosome"

Article Title: PPL2 Translesion Polymerase Is Essential for the Completion of Chromosomal DNA Replication in the African Trypanosome

Journal: Molecular Cell

doi: 10.1016/j.molcel.2013.10.034

TbPPL1 and TbPPL2 Are DNA-Dependent Translesion DNA Polymerases (A) DNA synthesis by His-tagged TbPPL1 and TbPPL2. Primer template substrate (20 nM) containing a 5′ fluorescent label on the primer strand and dNTPs (200 μM) were incubated without (−) or with wild-type (WT) TbPPL1 or TbPPL2 (50, 125, 250 nM) or catalytic (AxA) mutants (250 nM) for 30 min at 37°C. (B) Primer synthesis by TbPPL1 and TbPPL2. A single-stranded DNA template (500 nM) of variable sequence was incubated with either dNTPs or rNTPs (500 μM) and TbPPL1 or TbPPL2 (1 μM) for 2 hr at 37°C, and products were detected as described in Experimental Procedures . (C–F) DNA synthesis by TbPPL1 and TbPPL2 on templates containing a T-T pyrimidine (6-4) pyrimidone photoproduct (6-4 PP) (C), a T-T cis-syn cyclobutane pyrimidine dimer (CPD) (D), a 3-deaza 3-methyladenine (3dMeA) (E), and an 8-oxo-2′-deoxyguanosine (8-oxo-G) (F). (C), (E), and (F) contain substrates with primer termini 3′ of the templated lesion, thereby testing read-through of the lesion, while the primer in (D) contains two 3′ terminal dA residues annealed opposite the CPD, thereby testing extension from the lesion. Primer extensions performed as in (A) except with 125 nM TbPPL1 or TbPPL2 for 10, 20, and 30 min or just a single 30 min time point. As a control the archaeal family-B replicase Tgo-Pol exo − (100 nM) was used.
Figure Legend Snippet: TbPPL1 and TbPPL2 Are DNA-Dependent Translesion DNA Polymerases (A) DNA synthesis by His-tagged TbPPL1 and TbPPL2. Primer template substrate (20 nM) containing a 5′ fluorescent label on the primer strand and dNTPs (200 μM) were incubated without (−) or with wild-type (WT) TbPPL1 or TbPPL2 (50, 125, 250 nM) or catalytic (AxA) mutants (250 nM) for 30 min at 37°C. (B) Primer synthesis by TbPPL1 and TbPPL2. A single-stranded DNA template (500 nM) of variable sequence was incubated with either dNTPs or rNTPs (500 μM) and TbPPL1 or TbPPL2 (1 μM) for 2 hr at 37°C, and products were detected as described in Experimental Procedures . (C–F) DNA synthesis by TbPPL1 and TbPPL2 on templates containing a T-T pyrimidine (6-4) pyrimidone photoproduct (6-4 PP) (C), a T-T cis-syn cyclobutane pyrimidine dimer (CPD) (D), a 3-deaza 3-methyladenine (3dMeA) (E), and an 8-oxo-2′-deoxyguanosine (8-oxo-G) (F). (C), (E), and (F) contain substrates with primer termini 3′ of the templated lesion, thereby testing read-through of the lesion, while the primer in (D) contains two 3′ terminal dA residues annealed opposite the CPD, thereby testing extension from the lesion. Primer extensions performed as in (A) except with 125 nM TbPPL1 or TbPPL2 for 10, 20, and 30 min or just a single 30 min time point. As a control the archaeal family-B replicase Tgo-Pol exo − (100 nM) was used.

Techniques Used: DNA Synthesis, Incubation, Sequencing

4) Product Images from "Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach"

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach

Journal: Antiviral research

doi: 10.1016/j.antiviral.2008.05.010

Assay to distinguish polymerase and processive inhibitors of vaccinia DNA synthesis. (A) The model depicts the uniform incorporation of the DIG-dU on a template by E9 polymerase under low salt conditions. Under low salt conditions, E9 incorporates dNTPs along the DNA template. (B) NCI hit compounds were analyzed on the uniform tempate in the presence of E9 alone under low salt conditions to identify polymerase inhibitors. (C) The model depicts incorporation of the label DIG-dUTP on the distal end of the template by the triad (A20, D4, E9) under high salt conditions. This template was designed with all the adenines near the biotinylated end to direct incorporation of the DIG label towards the 3′ of the growing strand. Under high salt conditions, E9 requires A20 and D4 to accomplish processive DNA synthesis. (D) NCI hit compounds were analyzed on the distal tempate in the presence of the A20, D4 and E9 triad under high salt conditions to identify processivity inhibitors. Compounds that block E9 polymerase activity also blocked processivity. For both A and C, the template 5′ end is biotinylated for attachment to the streptavidin-coated plates.
Figure Legend Snippet: Assay to distinguish polymerase and processive inhibitors of vaccinia DNA synthesis. (A) The model depicts the uniform incorporation of the DIG-dU on a template by E9 polymerase under low salt conditions. Under low salt conditions, E9 incorporates dNTPs along the DNA template. (B) NCI hit compounds were analyzed on the uniform tempate in the presence of E9 alone under low salt conditions to identify polymerase inhibitors. (C) The model depicts incorporation of the label DIG-dUTP on the distal end of the template by the triad (A20, D4, E9) under high salt conditions. This template was designed with all the adenines near the biotinylated end to direct incorporation of the DIG label towards the 3′ of the growing strand. Under high salt conditions, E9 requires A20 and D4 to accomplish processive DNA synthesis. (D) NCI hit compounds were analyzed on the distal tempate in the presence of the A20, D4 and E9 triad under high salt conditions to identify processivity inhibitors. Compounds that block E9 polymerase activity also blocked processivity. For both A and C, the template 5′ end is biotinylated for attachment to the streptavidin-coated plates.

Techniques Used: DNA Synthesis, Blocking Assay, Activity Assay

5) Product Images from "Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens"

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl391

Primer blocking by di-deoxytermination. Well 1 contains 100 bp molecular-size standard (Roche) for Tfl and DVent (exo-) test sets and is empty for the rest of the enzyme sets. Wells 2–11 are the same for each enzyme tested and contain PCR product generated after supplementing 20% of the blocking reaction with fresh Taq and 10 mM dNTPs. Well 2, blocked with 10 mM ddNTPs and 10 mM dNTPs; Well 3, blocked with 10 mM ddNTPs and 5 mM dNTPs; Well 4, blocked with 10 mM ddNTPs and 1 mM dNTPs; Well 5, blocked with 10 mM ddNTPs and 0.1mM dNTPs; Well 6, blocked with 10 mM ddNTPs and 5 mM dATP and dCTP; Well 7, blocked with 10 mM ddNTPs and 5 mM dGTP and dTTP; Well 8, blocked with10 mM ddNTPs; Well 9, no ddNTPs in the blocking step; Well 10, no dNTPs in the blocking step Well 11, no primers or template.
Figure Legend Snippet: Primer blocking by di-deoxytermination. Well 1 contains 100 bp molecular-size standard (Roche) for Tfl and DVent (exo-) test sets and is empty for the rest of the enzyme sets. Wells 2–11 are the same for each enzyme tested and contain PCR product generated after supplementing 20% of the blocking reaction with fresh Taq and 10 mM dNTPs. Well 2, blocked with 10 mM ddNTPs and 10 mM dNTPs; Well 3, blocked with 10 mM ddNTPs and 5 mM dNTPs; Well 4, blocked with 10 mM ddNTPs and 1 mM dNTPs; Well 5, blocked with 10 mM ddNTPs and 0.1mM dNTPs; Well 6, blocked with 10 mM ddNTPs and 5 mM dATP and dCTP; Well 7, blocked with 10 mM ddNTPs and 5 mM dGTP and dTTP; Well 8, blocked with10 mM ddNTPs; Well 9, no ddNTPs in the blocking step; Well 10, no dNTPs in the blocking step Well 11, no primers or template.

Techniques Used: Blocking Assay, Polymerase Chain Reaction, Generated

6) Product Images from "Novel Aptamer Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase"

Article Title: Novel Aptamer Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase

Journal: Oligonucleotides

doi: 10.1089/oli.2008.0103

( A and B ) Inhibition assay showing that 37 NT SELEX is a potent inhibitor of HIV-RT primer extension. Shown is a schematic representation of the primer-template used in the assays ( A ) along with a graph ( B ) of extended primer vs. time in the presence of various amounts of 37 NT SELEX or 37 NT Random aptamers (as indicated). Each aptamer contained a 3′ terminal dideoxy G residue in place of the normal G. The assay was conducted by incubating HIV-RT (0.25 nM) with the primer-template (5′ γ- 32 P end-labeled primer, 50 nM) and aptamers, then initiating reactions with dNTPs and removing aliquots at the indicated times. The samples were run on a gel and quantified as described in Materials and Methods.
Figure Legend Snippet: ( A and B ) Inhibition assay showing that 37 NT SELEX is a potent inhibitor of HIV-RT primer extension. Shown is a schematic representation of the primer-template used in the assays ( A ) along with a graph ( B ) of extended primer vs. time in the presence of various amounts of 37 NT SELEX or 37 NT Random aptamers (as indicated). Each aptamer contained a 3′ terminal dideoxy G residue in place of the normal G. The assay was conducted by incubating HIV-RT (0.25 nM) with the primer-template (5′ γ- 32 P end-labeled primer, 50 nM) and aptamers, then initiating reactions with dNTPs and removing aliquots at the indicated times. The samples were run on a gel and quantified as described in Materials and Methods.

Techniques Used: Inhibition, Labeling

Related Articles

Amplification:

Article Title: Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China
Article Snippet: .. The two rounds of PCR amplification were both carried out in 25-μl reaction volumes containing 2.5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTPs, 0.5 μl (5 U) Taq DNA polymerase (Roche Molecular Systems), 1 μl (10 μM) each of sense (external or internal) and anti-sense primers, 2 μl template and 17 μl sterile water for 35 cycles of 94 °C for 30 s (additional 5 min for the first cycle), 53 °C for 30 s and 72 °C for 40 s (additional 10 min for the last cycle). .. Negative (diethyl pyrocarbonate water) and positive (positive swine liver) control samples were included during RNA extraction and first and second PCRs.

Article Title: InDel marker based genetic differentiation and genetic diversity in traditional rice (Oryza sativa L.) landraces of Chhattisgarh, India
Article Snippet: .. PCR amplification and electrophoresis analysis PCR reactions were performed in 10 μl mixture containing 4 μl (2.5 ɳg/ μl) template DNA, 2 μl of 5x assay buffer, 2 mM MgCl2 (Promega, Madison, USA), 0.2 μM of each forward and reverse InDel primer, 200 μM dNTPs (Roche, Indianapolis, USA) and 1.5 U of Taq DNA polymerase (BRIT, Mumbai, India). .. PCR reactions were carried out in a thermal-cycler (Eppendorf, Hamburg, Germany).

Article Title: ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing
Article Snippet: .. Alternatively, amplification was performed in 384-well plates with 2 μl template DNA, 1 μl 10x buffer mix without MgCl2 (Roche Fast Start Kit), 0.8 μl 25 mM MgCl2 , 0.5 μl DMSO, 0.2 μl 10 mM dNTPs each (Roche Fast Start Kit), 0.1 μl Fast Start Taq Polymerase (5 U/μl) (Roche Fast Start Kit), 4.4 μl PCR grade water and 1 μl of target-specific primer mix. .. We used a thermal profile of 95 °C for 4 min followed by 35 cycles at 95 °C for 25 s, 57 °C for 30 s and 72 °C for 90 s, and a finishing step at 72 °C for 5 min. Amplicons belonging to one sample were pooled with an CyBi-Well Vario system (Analytik Jena AG, Jena, Germany) and 2 μl transferred to an 9 μl pre-aliquoted PCR master mix including 1 μl 10x buffer mix without MgCl2 (Roche Fast Start Kit), 1 μl 25 mM MgCl2 , 0.2 μl DMSO, 0.2 μl 10 mM dNTPs each (Roche Fast Start Kit), 0.1 μl Fast Start Taq Polymerase (5 U/μl) (Roche Fast Start Kit), 3.5 μl PCR grade water as well as 2 μl of barcode primers (2 μM equimolar mix of index 1 and index 2).

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: .. The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science). .. PCR was performed using the following cycles conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 55 s and ended with an extension step at 72 °C for 5 minutes; products were visualized by electrophoresis on 1.2% agarose gel.

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: The incubation conditions for the RT were 70°C for 30 s, 47°C for 30 s, and 70°C for 15 min. PCR amplification of the synthesized cDNA was monitored on-line using the LightCycler instrument (Roche Molecular Biochemicals). .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

DNA Synthesis:

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach
Article Snippet: .. The 60 μL DNA synthesis reaction mixture contained 100 mM (NH4 )2 SO4 , 20 mM Tris-HCl pH 7.4, 3 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) and 1 μL vaccinia lysate. .. The plates were incubated at 37 °C for 30 min. DNA synthesis was determined by quantitating incorporation of DIG-dUTP using a DIG detection ELISA kit (Roche Applied Science) using anti-digoxigenin-peroxidase (anti-DIG-POD) and its substrate 2, 2′-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS), and by measuring the absorbance at 405 nm on a microplate reader (Tecan Genious Pro, Grodig, Austria).

Synthesized:

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: The incubation conditions for the RT were 70°C for 30 s, 47°C for 30 s, and 70°C for 15 min. PCR amplification of the synthesized cDNA was monitored on-line using the LightCycler instrument (Roche Molecular Biochemicals). .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

Quantitative RT-PCR:

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: Paragraph title: Real-time RT-PCR. ... PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

SYBR Green Assay:

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase. ..

Incubation:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: .. Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl. ..

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants. .. All reactions were incubated at 37°C for 30 min, reactions were quenched with addition of 2× stop buffer (95% formamide, 0.09% xylene cyanol, 0.05% bromophenol blue, 200 nM competitor oligonucleotide) and boiled at 95°C for 5 min.

Article Title: The Kluyveromyces lactis ?-toxin targets tRNA anticodons
Article Snippet: Purified γ-toxin-GST protein was mixed with total or purified tRNA in 10 mM Tris-HCl, 10 mM MgCl2 , 50 mM NaCl, and 1 mM dithiothreitol (pH 7.5) and incubated for 10 min at 30°C. .. Extensions were performed for 5 min at 37°C in the presence of 20 μM dNTPs and 25 U of AMV reverse transcriptase (Roche Applied Science).

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach
Article Snippet: The 96-well microtiter streptavidin-coated plates (Streptawell plates, Roche Applied Science, Indianapolis, IN) were coated with 5 pmol/well of the P/T solution and incubated at 37 °C for 90 min. .. The 60 μL DNA synthesis reaction mixture contained 100 mM (NH4 )2 SO4 , 20 mM Tris-HCl pH 7.4, 3 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) and 1 μL vaccinia lysate.

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: The incubation conditions for the RT were 70°C for 30 s, 47°C for 30 s, and 70°C for 15 min. PCR amplification of the synthesized cDNA was monitored on-line using the LightCycler instrument (Roche Molecular Biochemicals). .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). .. After 10 min incubation at > 60°C, the beads are captured on a magnet rack (Qiagen) and the supernatant removed to a fresh tube taking care that in the process the temperature remains > 55°C.

Activity Assay:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: .. Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl. ..

Mass Spectrometry:

Article Title: Genetic Polymorphisms in Toll-Like Receptors among Pediatric Patients with Renal Parenchymal Infections of Different Clinical Severities
Article Snippet: Briefly, PCR was performed in a total volume of 25 µL containing 200 ng of genomic DNA, primers (25 pM each), dNTPs (0.2 mM), 1× Fast-Start PCR buffer (50 mM Tris-HCl, 10 mM KCl, 5 mM [NH4 ]2 SO4 , 2mM MgCl2 ; pH 8.3), 1 M betaine, and 1 U of Fast-Start Taq Polymerase (Roche Diagnostics, Basel, Switzerland). .. The reactions were carried out in a multiblock thermal cycler (Thermo Hybaid, Waltham, MA) with initial denaturation at 96°C for 1 min, followed by 50 cycles of 96°C for 15 s, 50°C for 15 s, 60°C for 100 s, and 96°C for 30 s. The reaction products were purified automatically by MAPIIA (Single-Strand DNA Binding Beads; Bruker) and analyzed by MALDI-TOF mass spectrometry (MS).

Modification:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: .. Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl. ..

Recombinase Polymerase Amplification:

Article Title: Nucleosomes influence multiple steps during replication initiation
Article Snippet: .. Upon completion of DDK phosphorylation, the DDK reaction mix was removed from the beads and the replication initiation/elongation step was carried out by adding the indicated amounts of the following proteins (0.5 pmol S-CDK, 0.1 pmol DDK, 0.5 pmol Sld3/7, 2.5 pmol Cdc45, 1 pmol Sld2, 1 pmol Dpb11, 2.5 pmol GINS, 80 fmol Mcm10, 0.93 pmol Polε, 1.25 pmol Polα and 1 pmol RPA) in 30 µl of replication-initiation buffer (25 mM HEPES-KOH [pH7.6], 12.5 mM MgAc, 300 mM KGlut, 20 μM creatine phosphate, 0.02% NP40, 0.04 mg/ml BSA, 10% Glycerol, 3 mM ATP, 40 μM dNTPs, 200 μM CTP/UTP/GTP, 1 mM DTT, 10 μCi [α-P32 ] dCTP, 2 μg creatine kinase, and 0.5X complete protease inhibitor [Roche]) to the DNA beads for 60 min at 25°C and 1250 rpm in a Thermomixer. ..

Sequencing:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: Terminal transferase assays The terminal transferase capability of wild-type PrimPol and its variants was studied using three types of synthetic DNA substrates: ds DNA (sequence 6 annealed to sequence 7 from Supplementary Table S2) and a primer template containing both single-stranded and double-stranded DNA interfaces (sequence 5 annealed to sequence 6; Supplementary Table S2). .. Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants.

Article Title: The Kluyveromyces lactis ?-toxin targets tRNA anticodons
Article Snippet: Extensions were performed for 5 min at 37°C in the presence of 20 μM dNTPs and 25 U of AMV reverse transcriptase (Roche Applied Science). .. The samples were precipitated and applied next to a sequencing ladder on a denaturing 6% polyacrylamide gel.

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: Paragraph title: Microbial DNA extraction, 16S ribosomal DNA (rDNA) library preparation and sequencing ... The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science).

Article Title: Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)
Article Snippet: The detected novel, coding variant in MAF was validated by Sanger sequencing using forward primer 5′-GGGGGTGTGTGTGTGAGC-3′ and reverse primer 5′-CTGGAGCTGGTGGCTGTT-3′. .. PCR reactions of 20 μl final volume consisting of 1X Coraload PCR buffer (Qiagen), 0.1 mM dNTPs (Roche Diagnostics, Basel, Switzerland), 0.5 μM each primer, 0.5U Hot Star Plus Taq Polymerase (Qiagen) and 40 ng of DNA was prepared.

Ligation:

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Protease Inhibitor:

Article Title: Nucleosomes influence multiple steps during replication initiation
Article Snippet: .. Upon completion of DDK phosphorylation, the DDK reaction mix was removed from the beads and the replication initiation/elongation step was carried out by adding the indicated amounts of the following proteins (0.5 pmol S-CDK, 0.1 pmol DDK, 0.5 pmol Sld3/7, 2.5 pmol Cdc45, 1 pmol Sld2, 1 pmol Dpb11, 2.5 pmol GINS, 80 fmol Mcm10, 0.93 pmol Polε, 1.25 pmol Polα and 1 pmol RPA) in 30 µl of replication-initiation buffer (25 mM HEPES-KOH [pH7.6], 12.5 mM MgAc, 300 mM KGlut, 20 μM creatine phosphate, 0.02% NP40, 0.04 mg/ml BSA, 10% Glycerol, 3 mM ATP, 40 μM dNTPs, 200 μM CTP/UTP/GTP, 1 mM DTT, 10 μCi [α-P32 ] dCTP, 2 μg creatine kinase, and 0.5X complete protease inhibitor [Roche]) to the DNA beads for 60 min at 25°C and 1250 rpm in a Thermomixer. ..

Generated:

Article Title: Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)
Article Snippet: The library was generated with the Ion AmpliSeq library kit version 2.0 (Life Technologies, California, USA) and custom Ion Ampliseq primers according to the manufacturer’s protocols, and was sequenced on an Ion Torrent Personal Genome Machine using the Ion PGM Sequencing 200 Kit v2 and an Ion 318 chip (Life Technologies). .. PCR reactions of 20 μl final volume consisting of 1X Coraload PCR buffer (Qiagen), 0.1 mM dNTPs (Roche Diagnostics, Basel, Switzerland), 0.5 μM each primer, 0.5U Hot Star Plus Taq Polymerase (Qiagen) and 40 ng of DNA was prepared.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China
Article Snippet: Paragraph title: RNA extraction and nested reverse transcription polymerase chain reaction (RT-nPCR) ... The two rounds of PCR amplification were both carried out in 25-μl reaction volumes containing 2.5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTPs, 0.5 μl (5 U) Taq DNA polymerase (Roche Molecular Systems), 1 μl (10 μM) each of sense (external or internal) and anti-sense primers, 2 μl template and 17 μl sterile water for 35 cycles of 94 °C for 30 s (additional 5 min for the first cycle), 53 °C for 30 s and 72 °C for 40 s (additional 10 min for the last cycle).

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: The RT-PCR was done in two steps using rTth DNA polymerase (Applied Biosystems) in both steps. .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

Binding Assay:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl. .. The primer synthesis/labelling enzymatic reactions were terminated by adding 450 μl of binding-washing (B-W) buffer (10 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM EDTA).

Article Title: Genetic Polymorphisms in Toll-Like Receptors among Pediatric Patients with Renal Parenchymal Infections of Different Clinical Severities
Article Snippet: Briefly, PCR was performed in a total volume of 25 µL containing 200 ng of genomic DNA, primers (25 pM each), dNTPs (0.2 mM), 1× Fast-Start PCR buffer (50 mM Tris-HCl, 10 mM KCl, 5 mM [NH4 ]2 SO4 , 2mM MgCl2 ; pH 8.3), 1 M betaine, and 1 U of Fast-Start Taq Polymerase (Roche Diagnostics, Basel, Switzerland). .. The reactions were carried out in a multiblock thermal cycler (Thermo Hybaid, Waltham, MA) with initial denaturation at 96°C for 1 min, followed by 50 cycles of 96°C for 15 s, 50°C for 15 s, 60°C for 100 s, and 96°C for 30 s. The reaction products were purified automatically by MAPIIA (Single-Strand DNA Binding Beads; Bruker) and analyzed by MALDI-TOF mass spectrometry (MS).

DNA Extraction:

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: Paragraph title: Microbial DNA extraction, 16S ribosomal DNA (rDNA) library preparation and sequencing ... The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science).

Fluorescence:

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase. .. The temperature transition rate was set to 20°C/s, and fluorescence was monitored at the end of each extension.

Magnetic Beads:

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). .. Removal of biotinylated products : The cleaned product is heated to 95°C, and 50 μl of streptavidin-coated magnetic beads were added (SPHERO™ Streptavidin Magnetic Particles from Spherotech, Inc., Libertyville, IL).

Isolation:

Article Title: Nucleosomes influence multiple steps during replication initiation
Article Snippet: Upon completion of helicase loading, the DNA beads were isolated and the supernatant was removed. .. Upon completion of DDK phosphorylation, the DDK reaction mix was removed from the beads and the replication initiation/elongation step was carried out by adding the indicated amounts of the following proteins (0.5 pmol S-CDK, 0.1 pmol DDK, 0.5 pmol Sld3/7, 2.5 pmol Cdc45, 1 pmol Sld2, 1 pmol Dpb11, 2.5 pmol GINS, 80 fmol Mcm10, 0.93 pmol Polε, 1.25 pmol Polα and 1 pmol RPA) in 30 µl of replication-initiation buffer (25 mM HEPES-KOH [pH7.6], 12.5 mM MgAc, 300 mM KGlut, 20 μM creatine phosphate, 0.02% NP40, 0.04 mg/ml BSA, 10% Glycerol, 3 mM ATP, 40 μM dNTPs, 200 μM CTP/UTP/GTP, 1 mM DTT, 10 μCi [α-P32 ] dCTP, 2 μg creatine kinase, and 0.5X complete protease inhibitor [Roche]) to the DNA beads for 60 min at 25°C and 1250 rpm in a Thermomixer.

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Labeling:

Article Title: PPL2 Translesion Polymerase Is Essential for the Completion of Chromosomal DNA Replication in the African Trypanosome
Article Snippet: Briefly, a typical priming reaction contains 10 mM Bis-Tris-Propane-HCl (pH 7), 10 mM MgCl2 , 50 mM NaCl, 500 nM DNA template (with a 5′ biotin moiety; see ), 500 μM dNTPs (Roche) or rNTPs (Invitrogen), and 1 μM recombinant TbPPL1 or TbPPL2. .. The reaction products are labeled using 15 μM FAM-6-dATP (Jena-Biosciences) and 0.2 U of klenow-Taq.

Purification:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl. .. Following the primer synthesis reaction, the reaction mixture was supplemented with 0.2 U of Klenow Taq (purified as in Engelke et al. ( )) and 15 μM FAM-6-dATP (Jena-Biosciences), incubated at 37°C for 45 min to allow fluorescent labelling of de novo synthesised primers.

Article Title: The Kluyveromyces lactis ?-toxin targets tRNA anticodons
Article Snippet: Purified γ-toxin-GST protein was mixed with total or purified tRNA in 10 mM Tris-HCl, 10 mM MgCl2 , 50 mM NaCl, and 1 mM dithiothreitol (pH 7.5) and incubated for 10 min at 30°C. .. Extensions were performed for 5 min at 37°C in the presence of 20 μM dNTPs and 25 U of AMV reverse transcriptase (Roche Applied Science).

Article Title: PPL2 Translesion Polymerase Is Essential for the Completion of Chromosomal DNA Replication in the African Trypanosome
Article Snippet: Briefly, a typical priming reaction contains 10 mM Bis-Tris-Propane-HCl (pH 7), 10 mM MgCl2 , 50 mM NaCl, 500 nM DNA template (with a 5′ biotin moiety; see ), 500 μM dNTPs (Roche) or rNTPs (Invitrogen), and 1 μM recombinant TbPPL1 or TbPPL2. .. The labeled products are purified using the 5′ biotin moiety of the template, boiled to liberate the FAM-labeled primers, and then resolved on a 15% polyacrylamide/7M urea gel before fluorescent detection.

Article Title: Genetic Polymorphisms in Toll-Like Receptors among Pediatric Patients with Renal Parenchymal Infections of Different Clinical Severities
Article Snippet: Briefly, PCR was performed in a total volume of 25 µL containing 200 ng of genomic DNA, primers (25 pM each), dNTPs (0.2 mM), 1× Fast-Start PCR buffer (50 mM Tris-HCl, 10 mM KCl, 5 mM [NH4 ]2 SO4 , 2mM MgCl2 ; pH 8.3), 1 M betaine, and 1 U of Fast-Start Taq Polymerase (Roche Diagnostics, Basel, Switzerland). .. The reaction comprised initiation at 95°C for 5 min, followed by 40 cycles of 95°C for 45 s, 50°C for 45 s, and 60°C for 45 s, with a final extension at 52°C for 10 min. Unincorporated dNTPs and primers were removed automatically by MAPIIA (GenePure PCR Purification System; Bruker, Bremen, Germany).

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science). .. After a purification step with Agencourt AMPure XP (Beckman Coulter Inc), the amplicons were indexed with 10 subsequent cycles of PCR using the Nextera XT Index Kit (Illumina).

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: The ligation products are purified to remove the T4 ligase and buffer with QIAquick nucleotide removal column and eluted in 55 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Polymerase Chain Reaction:

Article Title: Genetic Polymorphisms in Toll-Like Receptors among Pediatric Patients with Renal Parenchymal Infections of Different Clinical Severities
Article Snippet: .. Briefly, PCR was performed in a total volume of 25 µL containing 200 ng of genomic DNA, primers (25 pM each), dNTPs (0.2 mM), 1× Fast-Start PCR buffer (50 mM Tris-HCl, 10 mM KCl, 5 mM [NH4 ]2 SO4 , 2mM MgCl2 ; pH 8.3), 1 M betaine, and 1 U of Fast-Start Taq Polymerase (Roche Diagnostics, Basel, Switzerland). .. The reaction comprised initiation at 95°C for 5 min, followed by 40 cycles of 95°C for 45 s, 50°C for 45 s, and 60°C for 45 s, with a final extension at 52°C for 10 min. Unincorporated dNTPs and primers were removed automatically by MAPIIA (GenePure PCR Purification System; Bruker, Bremen, Germany).

Article Title: Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China
Article Snippet: .. The two rounds of PCR amplification were both carried out in 25-μl reaction volumes containing 2.5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTPs, 0.5 μl (5 U) Taq DNA polymerase (Roche Molecular Systems), 1 μl (10 μM) each of sense (external or internal) and anti-sense primers, 2 μl template and 17 μl sterile water for 35 cycles of 94 °C for 30 s (additional 5 min for the first cycle), 53 °C for 30 s and 72 °C for 40 s (additional 10 min for the last cycle). .. Negative (diethyl pyrocarbonate water) and positive (positive swine liver) control samples were included during RNA extraction and first and second PCRs.

Article Title: InDel marker based genetic differentiation and genetic diversity in traditional rice (Oryza sativa L.) landraces of Chhattisgarh, India
Article Snippet: .. PCR amplification and electrophoresis analysis PCR reactions were performed in 10 μl mixture containing 4 μl (2.5 ɳg/ μl) template DNA, 2 μl of 5x assay buffer, 2 mM MgCl2 (Promega, Madison, USA), 0.2 μM of each forward and reverse InDel primer, 200 μM dNTPs (Roche, Indianapolis, USA) and 1.5 U of Taq DNA polymerase (BRIT, Mumbai, India). .. PCR reactions were carried out in a thermal-cycler (Eppendorf, Hamburg, Germany).

Article Title: ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing
Article Snippet: .. Alternatively, amplification was performed in 384-well plates with 2 μl template DNA, 1 μl 10x buffer mix without MgCl2 (Roche Fast Start Kit), 0.8 μl 25 mM MgCl2 , 0.5 μl DMSO, 0.2 μl 10 mM dNTPs each (Roche Fast Start Kit), 0.1 μl Fast Start Taq Polymerase (5 U/μl) (Roche Fast Start Kit), 4.4 μl PCR grade water and 1 μl of target-specific primer mix. .. We used a thermal profile of 95 °C for 4 min followed by 35 cycles at 95 °C for 25 s, 57 °C for 30 s and 72 °C for 90 s, and a finishing step at 72 °C for 5 min. Amplicons belonging to one sample were pooled with an CyBi-Well Vario system (Analytik Jena AG, Jena, Germany) and 2 μl transferred to an 9 μl pre-aliquoted PCR master mix including 1 μl 10x buffer mix without MgCl2 (Roche Fast Start Kit), 1 μl 25 mM MgCl2 , 0.2 μl DMSO, 0.2 μl 10 mM dNTPs each (Roche Fast Start Kit), 0.1 μl Fast Start Taq Polymerase (5 U/μl) (Roche Fast Start Kit), 3.5 μl PCR grade water as well as 2 μl of barcode primers (2 μM equimolar mix of index 1 and index 2).

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: .. The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science). .. PCR was performed using the following cycles conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 55 s and ended with an extension step at 72 °C for 5 minutes; products were visualized by electrophoresis on 1.2% agarose gel.

Article Title: Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)
Article Snippet: .. PCR reactions of 20 μl final volume consisting of 1X Coraload PCR buffer (Qiagen), 0.1 mM dNTPs (Roche Diagnostics, Basel, Switzerland), 0.5 μM each primer, 0.5U Hot Star Plus Taq Polymerase (Qiagen) and 40 ng of DNA was prepared. .. Final concentrating of Mg2+ was adjusted to 2.5 mM by adding the required amount of Mgcl2 (Qiagen).

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase. ..

Blocking Assay:

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). .. The product is purified with QIAquick nucleotide removal kit to remove the excess ddNTPs and eluted in 100 μl of 10 mM Tris (pH 8).

Chromatin Immunoprecipitation:

Article Title: Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)
Article Snippet: The library was generated with the Ion AmpliSeq library kit version 2.0 (Life Technologies, California, USA) and custom Ion Ampliseq primers according to the manufacturer’s protocols, and was sequenced on an Ion Torrent Personal Genome Machine using the Ion PGM Sequencing 200 Kit v2 and an Ion 318 chip (Life Technologies). .. PCR reactions of 20 μl final volume consisting of 1X Coraload PCR buffer (Qiagen), 0.1 mM dNTPs (Roche Diagnostics, Basel, Switzerland), 0.5 μM each primer, 0.5U Hot Star Plus Taq Polymerase (Qiagen) and 40 ng of DNA was prepared.

Enzyme-linked Immunosorbent Assay:

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach
Article Snippet: The 60 μL DNA synthesis reaction mixture contained 100 mM (NH4 )2 SO4 , 20 mM Tris-HCl pH 7.4, 3 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) and 1 μL vaccinia lysate. .. The plates were incubated at 37 °C for 30 min. DNA synthesis was determined by quantitating incorporation of DIG-dUTP using a DIG detection ELISA kit (Roche Applied Science) using anti-digoxigenin-peroxidase (anti-DIG-POD) and its substrate 2, 2′-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS), and by measuring the absorbance at 405 nm on a microplate reader (Tecan Genious Pro, Grodig, Austria).

RNA Extraction:

Article Title: Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China
Article Snippet: Paragraph title: RNA extraction and nested reverse transcription polymerase chain reaction (RT-nPCR) ... The two rounds of PCR amplification were both carried out in 25-μl reaction volumes containing 2.5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTPs, 0.5 μl (5 U) Taq DNA polymerase (Roche Molecular Systems), 1 μl (10 μM) each of sense (external or internal) and anti-sense primers, 2 μl template and 17 μl sterile water for 35 cycles of 94 °C for 30 s (additional 5 min for the first cycle), 53 °C for 30 s and 72 °C for 40 s (additional 10 min for the last cycle).

Recombinant:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: .. Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants. .. All reactions were incubated at 37°C for 30 min, reactions were quenched with addition of 2× stop buffer (95% formamide, 0.09% xylene cyanol, 0.05% bromophenol blue, 200 nM competitor oligonucleotide) and boiled at 95°C for 5 min.

Article Title: PPL2 Translesion Polymerase Is Essential for the Completion of Chromosomal DNA Replication in the African Trypanosome
Article Snippet: .. Briefly, a typical priming reaction contains 10 mM Bis-Tris-Propane-HCl (pH 7), 10 mM MgCl2 , 50 mM NaCl, 500 nM DNA template (with a 5′ biotin moiety; see ), 500 μM dNTPs (Roche) or rNTPs (Invitrogen), and 1 μM recombinant TbPPL1 or TbPPL2. .. The reaction products are labeled using 15 μM FAM-6-dATP (Jena-Biosciences) and 0.2 U of klenow-Taq.

Agarose Gel Electrophoresis:

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science). .. PCR was performed using the following cycles conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 55 s and ended with an extension step at 72 °C for 5 minutes; products were visualized by electrophoresis on 1.2% agarose gel.

In Vitro:

Article Title: The Kluyveromyces lactis ?-toxin targets tRNA anticodons
Article Snippet: Paragraph title: Characterization of tRNAs treated with γ-toxin in vitro ... Extensions were performed for 5 min at 37°C in the presence of 20 μM dNTPs and 25 U of AMV reverse transcriptase (Roche Applied Science).

Electrophoresis:

Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol
Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants. .. Samples were resolved by electrophoresis as described for the primer extension assays.

Article Title: InDel marker based genetic differentiation and genetic diversity in traditional rice (Oryza sativa L.) landraces of Chhattisgarh, India
Article Snippet: .. PCR amplification and electrophoresis analysis PCR reactions were performed in 10 μl mixture containing 4 μl (2.5 ɳg/ μl) template DNA, 2 μl of 5x assay buffer, 2 mM MgCl2 (Promega, Madison, USA), 0.2 μM of each forward and reverse InDel primer, 200 μM dNTPs (Roche, Indianapolis, USA) and 1.5 U of Taq DNA polymerase (BRIT, Mumbai, India). .. PCR reactions were carried out in a thermal-cycler (Eppendorf, Hamburg, Germany).

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science). .. PCR was performed using the following cycles conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 55 s and ended with an extension step at 72 °C for 5 minutes; products were visualized by electrophoresis on 1.2% agarose gel.

Concentration Assay:

Article Title: Molecular detection of hepatitis E virus in sheep from southern Xinjiang, China
Article Snippet: The two rounds of PCR amplification were both carried out in 25-μl reaction volumes containing 2.5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTPs, 0.5 μl (5 U) Taq DNA polymerase (Roche Molecular Systems), 1 μl (10 μM) each of sense (external or internal) and anti-sense primers, 2 μl template and 17 μl sterile water for 35 cycles of 94 °C for 30 s (additional 5 min for the first cycle), 53 °C for 30 s and 72 °C for 40 s (additional 10 min for the last cycle). .. The reaction system and conditions used in our study were capable of detecting 2.5 ng HEV RNA in liver, which was determined using serial mixtures by adding tenfold dilutions of swine HEV RNA into the swine liver suspensions with the same concentration.

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach
Article Snippet: Control (DMSO, acyclovir, azidothymidine (AZT) and ethylenediamine tetracetic acid (EDTA)) and test compounds were individually added to the wells to a final concentration of 167 μM. .. The 60 μL DNA synthesis reaction mixture contained 100 mM (NH4 )2 SO4 , 20 mM Tris-HCl pH 7.4, 3 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) and 1 μL vaccinia lysate.

Article Title: Sex-related alterations of gut microbiota composition in the BTBR mouse model of autism spectrum disorder
Article Snippet: DNA concentration was measured fluorometrically using Qubit dsDNA BR assay kit (Invitrogen) and quality was assessed by spectrophotometric measurements with NanoDrop (ThermoFisher Scientific Inc). .. The V3–V4 regions of the 16S rDNA gene were amplified starting from 200 ng of DNA template in a reaction volume of 50 μL containg 1x Fast start High Fidelity Reaction Buffer, 5 μM of each primer, 0.2 nM of dNTPs, 3 mM MgCl2 , and 2 U FastStart High Fidelity PCR System (Roche Applied Science).

Article Title: Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori
Article Snippet: The RT reaction mixture volume was 10 μl, comprising 1× reverse transcriptase buffer (Applied Biosystems), 1 mM MnCl2 , a 0.2 mM concentration of each dNTP, 0.5 μM reverse primer, 4 mg of bovine serum albumin per ml, and 1 U of rTth DNA polymerase. .. PCR was carried out in 20-μl volumes containing 1× chelating buffer (Applied Biosystems), 2.5 mM MgCl2 , 0.2 mM dNTPs, a 30,000-fold diluted stock solution of SYBR Green I (Roche Molecular Biochemicals), and 1.25 U of rTth DNA polymerase.

High Throughput Screening Assay:

Article Title: Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach
Article Snippet: Paragraph title: 2.3 High-throughput screening for inhibitors of DNA synthesis using the rapid plate assay ... The 60 μL DNA synthesis reaction mixture contained 100 mM (NH4 )2 SO4 , 20 mM Tris-HCl pH 7.4, 3 mM MgCl2 , 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) and 1 μL vaccinia lysate.

Gel Extraction:

Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Article Snippet: MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Variant Assay:

Article Title: Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome)
Article Snippet: The detected novel, coding variant in MAF was validated by Sanger sequencing using forward primer 5′-GGGGGTGTGTGTGTGAGC-3′ and reverse primer 5′-CTGGAGCTGGTGGCTGTT-3′. .. PCR reactions of 20 μl final volume consisting of 1X Coraload PCR buffer (Qiagen), 0.1 mM dNTPs (Roche Diagnostics, Basel, Switzerland), 0.5 μM each primer, 0.5U Hot Star Plus Taq Polymerase (Qiagen) and 40 ng of DNA was prepared.

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    Roche fitc dntp
    Inhibition of F4/80 + cell apoptosis reduces CD8 + T cell response and increases fungal burden. Wild-type mice were treated daily with Boc-D-FMK apoptosis inhibitor or Z-FA-FMK control peptide or left untreated starting at the day of intratracheal inoculation of 2 × 10 5 live Histoplasma . (A) Lung cells isolated from infected mice at day 4 after infection were stained with TUNEL reagents containing <t>FITC-dNTP</t> and PE-anti-F4/80 antibodies. Apoptotic F4/80 + cells in the lungs were analyzed by flow cytometry. Data shown represent the means ± SD of the total numbers of F4/80 + cells determined for 3 mice used in 3 independent experiments (*, P
    Fitc Dntp, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    fitc dntp - by Bioz Stars, 2020-02
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    dntps  (Roche)
    96
    Roche dntps
    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
    Dntps, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 821 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntps/product/Roche
    Average 96 stars, based on 821 article reviews
    Price from $9.99 to $1999.99
    dntps - by Bioz Stars, 2020-02
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    Inhibition of F4/80 + cell apoptosis reduces CD8 + T cell response and increases fungal burden. Wild-type mice were treated daily with Boc-D-FMK apoptosis inhibitor or Z-FA-FMK control peptide or left untreated starting at the day of intratracheal inoculation of 2 × 10 5 live Histoplasma . (A) Lung cells isolated from infected mice at day 4 after infection were stained with TUNEL reagents containing FITC-dNTP and PE-anti-F4/80 antibodies. Apoptotic F4/80 + cells in the lungs were analyzed by flow cytometry. Data shown represent the means ± SD of the total numbers of F4/80 + cells determined for 3 mice used in 3 independent experiments (*, P

    Journal: Infection and Immunity

    Article Title: Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+ T Cells To Protect against Histoplasmosis ▿

    doi: 10.1128/IAI.05350-11

    Figure Lengend Snippet: Inhibition of F4/80 + cell apoptosis reduces CD8 + T cell response and increases fungal burden. Wild-type mice were treated daily with Boc-D-FMK apoptosis inhibitor or Z-FA-FMK control peptide or left untreated starting at the day of intratracheal inoculation of 2 × 10 5 live Histoplasma . (A) Lung cells isolated from infected mice at day 4 after infection were stained with TUNEL reagents containing FITC-dNTP and PE-anti-F4/80 antibodies. Apoptotic F4/80 + cells in the lungs were analyzed by flow cytometry. Data shown represent the means ± SD of the total numbers of F4/80 + cells determined for 3 mice used in 3 independent experiments (*, P

    Article Snippet: The sections were then treated with 0.1% Triton X-100 (Sigma-Aldrich) at 4°C for 5 min and incubated with a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture containing FITC-dNTP (Roche, Basel, Switzerland) at 37°C in the dark for 1 h. After being washed with PBS, the slides were stained with PE-anti-F4/80 or PE-anti-Gr-1.

    Techniques: Inhibition, Mouse Assay, Isolation, Infection, Staining, TUNEL Assay, Flow Cytometry, Cytometry

    iNOS deficiency reduces F4/80 + cell apoptosis and weakens CD8 + T cell response in pulmonary histoplasmosis. Wild-type and iNOS −/− mice were infected intratracheally with 2 × 10 5 live Histoplasma. (A) At day 5 after infection, lung cells were isolated and stained with PE-anti-F4/80 antibody and TUNEL reagents containing FITC-dNTP. F4/80 + TUNEL + apoptotic cells were analyzed by flow cytometry. Data shown represent the means ± SD of the total numbers of apoptotic F4/80 + cells determined for 3 mice used in 3 independent experiments. (B) At day 10 after infection, the mediastinal lymph nodes were harvested and cells were stained with PE-anti-IFN-γ and FITC-anti-CD4 or APC-anti-CD8 antibodies. Data shown represent the means ± SD of the percentages of IFN-γ-producing CD8 + or CD4 + T cells in the total CD8 + or CD4 + T cell population determined for 6 mice used in 3 independent experiments. Cells harvested from uninfected wild-type mice served as controls. The P values were obtained by comparing the results determined for pairs of groups (linked by a bracket) using Student's t test (*, P

    Journal: Infection and Immunity

    Article Title: Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+ T Cells To Protect against Histoplasmosis ▿

    doi: 10.1128/IAI.05350-11

    Figure Lengend Snippet: iNOS deficiency reduces F4/80 + cell apoptosis and weakens CD8 + T cell response in pulmonary histoplasmosis. Wild-type and iNOS −/− mice were infected intratracheally with 2 × 10 5 live Histoplasma. (A) At day 5 after infection, lung cells were isolated and stained with PE-anti-F4/80 antibody and TUNEL reagents containing FITC-dNTP. F4/80 + TUNEL + apoptotic cells were analyzed by flow cytometry. Data shown represent the means ± SD of the total numbers of apoptotic F4/80 + cells determined for 3 mice used in 3 independent experiments. (B) At day 10 after infection, the mediastinal lymph nodes were harvested and cells were stained with PE-anti-IFN-γ and FITC-anti-CD4 or APC-anti-CD8 antibodies. Data shown represent the means ± SD of the percentages of IFN-γ-producing CD8 + or CD4 + T cells in the total CD8 + or CD4 + T cell population determined for 6 mice used in 3 independent experiments. Cells harvested from uninfected wild-type mice served as controls. The P values were obtained by comparing the results determined for pairs of groups (linked by a bracket) using Student's t test (*, P

    Article Snippet: The sections were then treated with 0.1% Triton X-100 (Sigma-Aldrich) at 4°C for 5 min and incubated with a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture containing FITC-dNTP (Roche, Basel, Switzerland) at 37°C in the dark for 1 h. After being washed with PBS, the slides were stained with PE-anti-F4/80 or PE-anti-Gr-1.

    Techniques: Mouse Assay, Infection, Isolation, Staining, TUNEL Assay, Flow Cytometry, Cytometry

    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

    Article Snippet: Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl.

    Techniques: Activity Assay, Functional Assay

    Template-independent extension in the presence of manganese. Human PrimPol was incubated for 30 min with DNA substrate and each of the dNTPs in the presence of manganese. ( A ) Wild-type PrimPol was unable to extend from a ds DNA template with a blunt end, but could extend from a primer annealed to an overhanging template, even synthesising long tracts of homopolymers. ( B ) PrimPol ZF-KO could extend from an overhanging DNA template in the presence of manganese and, consistent with the wild-type, did not extend from a ds DNA substrate. The incorporation of 1 or 2 nucleotides of guanine or adenine opposite an overhanging template suggests that PrimPol ZF-KO incorporates in a low-fidelity template-dependent manner when incubated with overhanging DNA. ( C ) PrimPol 1–487 exhibited a highly similar terminal transferase activity spectrum to the PrimPol ZF-KO . In the presence of manganese, it incorporated bases opposite an overhang in a low fidelity, template-dependent manner. ( D ) PrimPol 1–354 also exhibited low fidelity extension of a primer annealed to an overhanging template in the presence of manganese.

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: Template-independent extension in the presence of manganese. Human PrimPol was incubated for 30 min with DNA substrate and each of the dNTPs in the presence of manganese. ( A ) Wild-type PrimPol was unable to extend from a ds DNA template with a blunt end, but could extend from a primer annealed to an overhanging template, even synthesising long tracts of homopolymers. ( B ) PrimPol ZF-KO could extend from an overhanging DNA template in the presence of manganese and, consistent with the wild-type, did not extend from a ds DNA substrate. The incorporation of 1 or 2 nucleotides of guanine or adenine opposite an overhanging template suggests that PrimPol ZF-KO incorporates in a low-fidelity template-dependent manner when incubated with overhanging DNA. ( C ) PrimPol 1–487 exhibited a highly similar terminal transferase activity spectrum to the PrimPol ZF-KO . In the presence of manganese, it incorporated bases opposite an overhang in a low fidelity, template-dependent manner. ( D ) PrimPol 1–354 also exhibited low fidelity extension of a primer annealed to an overhanging template in the presence of manganese.

    Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants.

    Techniques: Incubation, Activity Assay

    Polymerase activity and fidelity of human PrimPol. ( A ) Human PrimPol was incubated with dNTPs and substrate at 1, 3, 5 and 30 min time points. PrimPol was proficient at extending an undamaged oligonucleotide template using dNTPs. Human PrimPol did not require an intact zinc finger in order to carry out primer extension, as evidenced by the extension of primers by PrimPol ZF-KO and PrimPol 1–354 . PrimPol 1–487 that lacked the unstructured C-terminus of the protein was also polymerase proficient. PrimPol 1–354 exhibited a higher rate of polymerase activity compared to the other constructs. ( B ) Incorporation of nucleotides opposite two templating cytosine bases. PrimPol was incubated for 5 min with the DNA substrate and each of the dNTPs. All four of these PrimPol constructs inserted two guanine nucleotides opposite two cytosines in Watson–Crick base-pairing manner. PrimPol 1–354 could additionally incorporate a single adenine opposite the first cytosine.

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: Polymerase activity and fidelity of human PrimPol. ( A ) Human PrimPol was incubated with dNTPs and substrate at 1, 3, 5 and 30 min time points. PrimPol was proficient at extending an undamaged oligonucleotide template using dNTPs. Human PrimPol did not require an intact zinc finger in order to carry out primer extension, as evidenced by the extension of primers by PrimPol ZF-KO and PrimPol 1–354 . PrimPol 1–487 that lacked the unstructured C-terminus of the protein was also polymerase proficient. PrimPol 1–354 exhibited a higher rate of polymerase activity compared to the other constructs. ( B ) Incorporation of nucleotides opposite two templating cytosine bases. PrimPol was incubated for 5 min with the DNA substrate and each of the dNTPs. All four of these PrimPol constructs inserted two guanine nucleotides opposite two cytosines in Watson–Crick base-pairing manner. PrimPol 1–354 could additionally incorporate a single adenine opposite the first cytosine.

    Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants.

    Techniques: Activity Assay, Incubation, Construct

    Processivity of the polymerase activity of PrimPol. PrimPol was pre-incubated for 30 min at 37°C with an undamaged DNA primer-template substrate to allow PrimPol to bind to the DNA. The reaction was initiated by the addition of dNTPs and an excess of sonicated herring sperm DNA (trap) and time points taken at 15, 30, 60, 120 and 360 s. ( A ) After 360 s, wild-type PrimPol incorporated up to 4 nucleotides opposite the template but a significant fraction of enzyme incorporated only 1, 2 or 3 nucleotides (left panel). To confirm that the trap prevents polymerase extending from a second template, the trap was also added into the pre-incubation mix with PrimPol and the DNA substrate. This reaction was supplemented with dNTPs and there is no extension (right panel), thus successfully exhibiting the effectiveness of the trap. ( B ) PrimPol 1–354 also predominantly incorporates up to 4 nucleotides but there were fewer polymerases incorporating only 1, 2 or 3 nucleotides (left panel). The effectiveness of the trap was also successfully confirmed (right panel). ( C ) Percentage of PrimPol molecules incorporating at least n dNTPs for either full-length PrimPol or PrimPol 1–354 , calculated using Equation (1) .

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: Processivity of the polymerase activity of PrimPol. PrimPol was pre-incubated for 30 min at 37°C with an undamaged DNA primer-template substrate to allow PrimPol to bind to the DNA. The reaction was initiated by the addition of dNTPs and an excess of sonicated herring sperm DNA (trap) and time points taken at 15, 30, 60, 120 and 360 s. ( A ) After 360 s, wild-type PrimPol incorporated up to 4 nucleotides opposite the template but a significant fraction of enzyme incorporated only 1, 2 or 3 nucleotides (left panel). To confirm that the trap prevents polymerase extending from a second template, the trap was also added into the pre-incubation mix with PrimPol and the DNA substrate. This reaction was supplemented with dNTPs and there is no extension (right panel), thus successfully exhibiting the effectiveness of the trap. ( B ) PrimPol 1–354 also predominantly incorporates up to 4 nucleotides but there were fewer polymerases incorporating only 1, 2 or 3 nucleotides (left panel). The effectiveness of the trap was also successfully confirmed (right panel). ( C ) Percentage of PrimPol molecules incorporating at least n dNTPs for either full-length PrimPol or PrimPol 1–354 , calculated using Equation (1) .

    Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants.

    Techniques: Activity Assay, Incubation, Sonication

    PrimPol 1–354 can replicate through CPD and (6–4(PP)) lesions. ( A ) PrimPol 1–354 was incubated with a primer-template substrate in which the template contained a CPD lesion downstream of the primer-template junction in the presence of dNTPs. Time points were taken between 0.5 and 60 min. PrimPol 1–354 extends from the primer up to the CPD, before stalling, it will then added a base opposite the first thymine of the CPD and continue to extend until the end of the template (left panel). PrimPol 1–354 was then incubated with each dNTP to test which nucleotide it incorporates opposite a CPD (right panels). PrimPol 1–354 incorporated adenine opposite the first and second thymine of the CPD. ( B ) The polymerase domain of PrimPol can also perform TLS bypass of a 6–4(PP) lesion immediately downstream of the primer-template junction (left panel). PrimPol 1–354 incorporates either an adenine or cytosine nucleotide opposite the first thymine of the 6–4(PP) (right panels). If an adenine is incorporated opposite the first thymine, a cytosine or thymine is then incorporated opposite the second. If a cytosine is incorporated opposite the first thymine, an adenine, cytosine or thymine will be incorporated opposite the second thymine of the 6–4(PP).

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: PrimPol 1–354 can replicate through CPD and (6–4(PP)) lesions. ( A ) PrimPol 1–354 was incubated with a primer-template substrate in which the template contained a CPD lesion downstream of the primer-template junction in the presence of dNTPs. Time points were taken between 0.5 and 60 min. PrimPol 1–354 extends from the primer up to the CPD, before stalling, it will then added a base opposite the first thymine of the CPD and continue to extend until the end of the template (left panel). PrimPol 1–354 was then incubated with each dNTP to test which nucleotide it incorporates opposite a CPD (right panels). PrimPol 1–354 incorporated adenine opposite the first and second thymine of the CPD. ( B ) The polymerase domain of PrimPol can also perform TLS bypass of a 6–4(PP) lesion immediately downstream of the primer-template junction (left panel). PrimPol 1–354 incorporates either an adenine or cytosine nucleotide opposite the first thymine of the 6–4(PP) (right panels). If an adenine is incorporated opposite the first thymine, a cytosine or thymine is then incorporated opposite the second. If a cytosine is incorporated opposite the first thymine, an adenine, cytosine or thymine will be incorporated opposite the second thymine of the 6–4(PP).

    Article Snippet: Typical reactions were performed in 20 μl volume containing 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM NaCl, 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT, 20 nM DNA substrate, 200 μM dNTPs (Roche), with 100 nM recombinant human PrimPol or its variants.

    Techniques: Incubation