Structured Review

New England Biolabs dntps
The effect of SSBs on the primer extension activity of PrimPol. (A) Primer-template substrates (20 nM) and <t>dNTPs</t> (100 μM) were incubated with PrimPol (100 nM), either alone or with RPA (400 nM), mtSSB (200 nM) or <t>T4</t> SSB (400 nM), for increasing times (1, 3, 5, 10 min). In the presence of SSBs, full-length PrimPol's primer extension activity is severely impeded. The primer runs at the position indicated ‘ N ’, with full extension denoted by ‘ N + 77’. ‘C’ indicates the no enzyme control. For each gel, the 10-min time-point was quantified to identify the percentage of primers extended more than 1, 5, 10, 15, 20 or 25 bases. This quantification is shown on the right of the gels. (B) The primer extension activity of PrimPol 24–354 is also restricted in the presence of SSBs. Quantification of the 10-min time-point for each gel is again shown to the right of the gels. (C) Schematic representation of the inhibitory effect of SSBs on the primer extension activity of PrimPol.
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Images

1) Product Images from "Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins"

Article Title: Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku1321

The effect of SSBs on the primer extension activity of PrimPol. (A) Primer-template substrates (20 nM) and dNTPs (100 μM) were incubated with PrimPol (100 nM), either alone or with RPA (400 nM), mtSSB (200 nM) or T4 SSB (400 nM), for increasing times (1, 3, 5, 10 min). In the presence of SSBs, full-length PrimPol's primer extension activity is severely impeded. The primer runs at the position indicated ‘ N ’, with full extension denoted by ‘ N + 77’. ‘C’ indicates the no enzyme control. For each gel, the 10-min time-point was quantified to identify the percentage of primers extended more than 1, 5, 10, 15, 20 or 25 bases. This quantification is shown on the right of the gels. (B) The primer extension activity of PrimPol 24–354 is also restricted in the presence of SSBs. Quantification of the 10-min time-point for each gel is again shown to the right of the gels. (C) Schematic representation of the inhibitory effect of SSBs on the primer extension activity of PrimPol.
Figure Legend Snippet: The effect of SSBs on the primer extension activity of PrimPol. (A) Primer-template substrates (20 nM) and dNTPs (100 μM) were incubated with PrimPol (100 nM), either alone or with RPA (400 nM), mtSSB (200 nM) or T4 SSB (400 nM), for increasing times (1, 3, 5, 10 min). In the presence of SSBs, full-length PrimPol's primer extension activity is severely impeded. The primer runs at the position indicated ‘ N ’, with full extension denoted by ‘ N + 77’. ‘C’ indicates the no enzyme control. For each gel, the 10-min time-point was quantified to identify the percentage of primers extended more than 1, 5, 10, 15, 20 or 25 bases. This quantification is shown on the right of the gels. (B) The primer extension activity of PrimPol 24–354 is also restricted in the presence of SSBs. Quantification of the 10-min time-point for each gel is again shown to the right of the gels. (C) Schematic representation of the inhibitory effect of SSBs on the primer extension activity of PrimPol.

Techniques Used: Activity Assay, Incubation, Recombinase Polymerase Amplification

The effect of SSBs on the primase activity of human PrimPol. Single-stranded poly-dT templates (500 nM) were incubated with dNTPs or rNTPs (500 μM) and human PrimPol (1 μM), either alone or in the presence of RPA (8 μM), mtSSB (4 μM) or T4 SSB (8 μM) for 1 h at 37ºC (see ‘Materials and Methods’ for details). In the absence of SSBs PrimPol is capable of de novo primer synthesis using either dNTPs or rNTPs. However, when templates are coated with SSBs PrimPol is unable to synthesize primers. The schematic above represents how this inhibition is likely a result of RPA and mtSSB blocking PrimPol binding sites on the ssDNA.
Figure Legend Snippet: The effect of SSBs on the primase activity of human PrimPol. Single-stranded poly-dT templates (500 nM) were incubated with dNTPs or rNTPs (500 μM) and human PrimPol (1 μM), either alone or in the presence of RPA (8 μM), mtSSB (4 μM) or T4 SSB (8 μM) for 1 h at 37ºC (see ‘Materials and Methods’ for details). In the absence of SSBs PrimPol is capable of de novo primer synthesis using either dNTPs or rNTPs. However, when templates are coated with SSBs PrimPol is unable to synthesize primers. The schematic above represents how this inhibition is likely a result of RPA and mtSSB blocking PrimPol binding sites on the ssDNA.

Techniques Used: Activity Assay, Incubation, Recombinase Polymerase Amplification, Inhibition, Blocking Assay, Binding Assay

2) Product Images from "microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes"

Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku1188

Relative quantity of circularized MIP products. The y-axis shows the ‘percentage of unique reads’, with unique reads defined as those having distinct barcodes and arm sequence combinations. Since the number of unique reads increases as total number of reads increases, in order to normalize it, we randomly selected 2 million reads for each condition, counted the number of unique reads and calculated the percentage of unique reads. As the amount of gDNA or hybridization time increased, more unique reads were detected, indicating that more circularized product was obtained. A 1:500 gDNA:probe ratio and 10x dNTPs were used for all conditions.
Figure Legend Snippet: Relative quantity of circularized MIP products. The y-axis shows the ‘percentage of unique reads’, with unique reads defined as those having distinct barcodes and arm sequence combinations. Since the number of unique reads increases as total number of reads increases, in order to normalize it, we randomly selected 2 million reads for each condition, counted the number of unique reads and calculated the percentage of unique reads. As the amount of gDNA or hybridization time increased, more unique reads were detected, indicating that more circularized product was obtained. A 1:500 gDNA:probe ratio and 10x dNTPs were used for all conditions.

Techniques Used: Sequencing, Hybridization

Comparison of capture efficiencies under different conditions. The effects of varying ( A ) the gDNA:probe ratio, and ( B ) the amount of dNTPs are shown. Band intensity (∼200 bp, red arrows) is proportional to the amount of captured product on each of the capture parameters, because the number of PCR cycles was held constant at 26 cycles. The amount of captured products was saturated at 1:500 gDNA:probe ratio and 10x dNTPs. ( C ) and ( D ) Captured products were detected around 200 bp for all conditions, and only products in these bands were separated and used for further analysis.
Figure Legend Snippet: Comparison of capture efficiencies under different conditions. The effects of varying ( A ) the gDNA:probe ratio, and ( B ) the amount of dNTPs are shown. Band intensity (∼200 bp, red arrows) is proportional to the amount of captured product on each of the capture parameters, because the number of PCR cycles was held constant at 26 cycles. The amount of captured products was saturated at 1:500 gDNA:probe ratio and 10x dNTPs. ( C ) and ( D ) Captured products were detected around 200 bp for all conditions, and only products in these bands were separated and used for further analysis.

Techniques Used: Polymerase Chain Reaction

3) Product Images from "Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)"

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067558

PAGE electrophoresis of PEAR products. For dNTPs, lowercase letters (agct) represents natural dNTPs, and uppercase letters (AGCT) represents dNTPαSs. (A) PEAR products incorporating natural or dATPαS, dGTPαS, dCTPαS, dTTPαS: Lane 1: natural dNTPs; Lane 2: dATPαSs; Lane 3: No PspGI control; Lane 4: No Phusion DNA polymerase control; Lane 5: No dATP control; Lane 6∶10bp DNA ladder; Lane 7: dGTPαS; Lane 8: No PspGI control; Lane 9: No Phusion DNA polymerase control; Lane 10: No dCTP control; Lane 11: dCTPαSs; Lane 12: No PspGI control; Lane 13: No Phusion DNA polymerase control; Lane 14: No dCTP control; Lane 15: dTTPαSs; Lane 16: No PspGI control; Lane 17: No Phusion DNA polymerase control; Lane 18: No dTTP control; Lane 19∶10bp DNA ladder. (B) PEAR products incorporating one or two kind of dNTPαSs: Lane 1: natural dNTPs; Lane 2–5: one kind of dNTPαSs; Lane 6–8: two kind of dNTPαSs; Lane 9: No dNTPs control; Lane 10∶10bp DNA ladder; (C) Full digestion of PEAR products incorporating different dNTPs or dNTPαSs.
Figure Legend Snippet: PAGE electrophoresis of PEAR products. For dNTPs, lowercase letters (agct) represents natural dNTPs, and uppercase letters (AGCT) represents dNTPαSs. (A) PEAR products incorporating natural or dATPαS, dGTPαS, dCTPαS, dTTPαS: Lane 1: natural dNTPs; Lane 2: dATPαSs; Lane 3: No PspGI control; Lane 4: No Phusion DNA polymerase control; Lane 5: No dATP control; Lane 6∶10bp DNA ladder; Lane 7: dGTPαS; Lane 8: No PspGI control; Lane 9: No Phusion DNA polymerase control; Lane 10: No dCTP control; Lane 11: dCTPαSs; Lane 12: No PspGI control; Lane 13: No Phusion DNA polymerase control; Lane 14: No dCTP control; Lane 15: dTTPαSs; Lane 16: No PspGI control; Lane 17: No Phusion DNA polymerase control; Lane 18: No dTTP control; Lane 19∶10bp DNA ladder. (B) PEAR products incorporating one or two kind of dNTPαSs: Lane 1: natural dNTPs; Lane 2–5: one kind of dNTPαSs; Lane 6–8: two kind of dNTPαSs; Lane 9: No dNTPs control; Lane 10∶10bp DNA ladder; (C) Full digestion of PEAR products incorporating different dNTPs or dNTPαSs.

Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis

4) Product Images from "Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases"

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

Journal: Nucleic Acid Therapeutics

doi: 10.1089/nat.2014.0513

Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.
Figure Legend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

Related Articles

Clone Assay:

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. HIV-1 NC was cloned into pET28b, between NdeI and XhoI, to give an N terminal His-tagged construct.

Centrifugation:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: The fragments were precipitated with 70 mM sodium actetate (Life Technologies), 40 μg glycogen (Life Technologies) and 70% ethanol at −80°C for 1 hour followed by centrifugation and washing with 70% ethanol. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Amplification:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. Libraries were amplified for 13–16 cycles (up to 21 cycles for size-selected libraries) with primers that add the full Illumina sequencing adaptor sequences (an optimized variant of TruSeq adaptors, ) and NEBNext 2 × PCR Master Mix (New England Biolabs), using an aliquot of the sample to determine the optimal number of cycles as in ref. .

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Each entire sample was input into the Ambion WT Expression Kit (Life Technologies) to perform double stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
Article Snippet: Paragraph title: Rolling circle amplification of DNA from yeast artificial chromosomes ... Upon cooling, 15 μl of reaction buffer, 1 μl enzyme and 2 μl of 10 mM dNTPs (New England Biolabs) were added, and the reaction was incubated for 24 h at 30°C.

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: Each primer was uniquely modified on the 5′end with a 10-bp multiplex identifier (MID; Roche Diagnostics Technical Bulletin TCB No.005–2009), and samples were amplified using a unique combination of forward and reverse MID-labelled primers, which allowed recovery of the amplicons per individual after demultiplexing. .. PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes).

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: .. Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template. .. PCR was performed as follows: denaturation at 95°C for 50 sec, primer annealing at 65°C for 50 sec, and extension at 68°C for 7 min with a final extension for 5 min at 72°C.

Positive Control:

Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target
Article Snippet: Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB). .. For streptavidin experiments, positive control particles were made by labeling particles with 1/10 molar ratio 5′ biotinylated complementary oligo (see Supplementary Table S1 for oligo sequences).

Synthesized:

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The recognition site (R) of PspGI is CCWGG, where W = A or T. Synthetic ODNs, including a target (X ) and a probe (P ), are synthesized by Integrated DNA Technologies, Inc. and purified by HPLC.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

Construct:

Article Title: Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
Article Snippet: Rolling circle amplification was conducted using reagents from the TempliPhi Large Construct Kit (GE Healthcare). .. Upon cooling, 15 μl of reaction buffer, 1 μl enzyme and 2 μl of 10 mM dNTPs (New England Biolabs) were added, and the reaction was incubated for 24 h at 30°C.

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. HIV-1 NC was cloned into pET28b, between NdeI and XhoI, to give an N terminal His-tagged construct.

SYBR Green Assay:

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA). .. Each real-time PCR reaction consisted of 6 µl of diluted reverse transcribed product, 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 50 nM of forward and reverse primers.

Incubation:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: 3 μg of random hexamer (Life Technologies) was added to the fragmented, purified cRNA and incubated at 70°C for 10 min to anneal the primer. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes
Article Snippet: .. Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C. .. Next, 0.5 μl of Exonuclease I (NEB® Inc) and 0.5 μl of Exonuclease III (NEB® Inc) were used to remove linear DNA fragments at 37°C, and a 5-min incubation at 95°C followed for deactivation.

Article Title: Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
Article Snippet: .. Upon cooling, 15 μl of reaction buffer, 1 μl enzyme and 2 μl of 10 mM dNTPs (New England Biolabs) were added, and the reaction was incubated for 24 h at 30°C. .. To release the recoded DNA as a linear construct, the amplified DNA was diluted with 40 μl water, then 8 μl of 10× FastDigest buffer and 2 μl LguI restriction enzyme (Thermo Fisher) was added.

Diffusion-based Assay:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Plugs were washed four times in 1 mL TE at 4 °C. ssDNA fragments were recovered from the agarose plugs by thermal denaturation for 8 min at 95 °C followed by 2 min re-solidification on ice and passive diffusion into 140 μL TE per plug piece for ~16 h at room temperature with rotation. .. Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min.

Expressing:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Each entire sample was input into the Ambion WT Expression Kit (Life Technologies) to perform double stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. This expression plasmid was transformed into BL21 cells which were grown in Luria-Bertani medium until OD600 = 0.7, at which time expression was induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were supplemented with 50 µM Zinc acetate.

Modification:

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: Each primer was uniquely modified on the 5′end with a 10-bp multiplex identifier (MID; Roche Diagnostics Technical Bulletin TCB No.005–2009), and samples were amplified using a unique combination of forward and reverse MID-labelled primers, which allowed recovery of the amplicons per individual after demultiplexing. .. PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes).

Transformation Assay:

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. This expression plasmid was transformed into BL21 cells which were grown in Luria-Bertani medium until OD600 = 0.7, at which time expression was induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were supplemented with 50 µM Zinc acetate.

Recombinase Polymerase Amplification:

Article Title: Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins
Article Snippet: Extensions were carried out at 37°C in 20 μl volumes containing; 100 nM of the assayed polymerase (or 3U/ml of T4 Pol), 20 nM primer-template substrate, 100 μM dNTPs (NEB), 50 mM Potassium acetate, 20 mM Tris-acetate pH 7.9, 10 mM Magnesium acetate, 1 mM DTT and 2 μg bovine serum albumin (BSA; NEB). .. For assays using SSBs, DNA templates were pre-incubated on ice with 200 nM mtSSB, 400 nM RPA, or 400 nM T4 SSB, before the addition of enzymes.

Derivative Assay:

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The sequence of X is: TG T AAA CAT CCT CGA CTG GAA G , which is derived from human microRNA hsa-miR-30a.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. The sequence of X is 5′-TGT AAA CAT CCT CGA CTG GAA G-3′, which is derived from human microRNA hsa-miR-30a.

High Performance Liquid Chromatography:

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The recognition site (R) of PspGI is CCWGG, where W = A or T. Synthetic ODNs, including a target (X ) and a probe (P ), are synthesized by Integrated DNA Technologies, Inc. and purified by HPLC.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

Polymerase Chain Reaction:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: The eluates were diluted 20-fold in autoclaved MilliQ water, and 1 μl of each eluate was used as a template in a separate 50-μl PCR containing 500 n m YD-tag-F and YD-tag-R primers, 200 μ m ). .. The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer.

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: .. Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. The extension reactions were purified with a QIAQUICK PCR cleanup kit (Qiagen) and concentrated to 10–20 μL per sample.

Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes
Article Snippet: Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C. .. The post-amplification reaction used 1 μl of hybridized template, 10 μl of QIAGEN Multiplex PCR Master Mix (QIAGEN), 8 μl of dH2 O with 1 μl each of Capture_AmpF and Capture_AmpR primers (Supplementary Table S1).

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9
Article Snippet: Paragraph title: Junction capture PCR ... Twenty microliter of reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo).

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes). .. PCR amplicons were pooled and prepared for 150-bp paired-end Illumina MiSeq (Illumina, Inc., San Diego, CA, USA) sequencing using the vendor’s TruSeq library protocol.

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: The polymerase chain reaction (PCR) was applied to identify opisthorchiid species using 3 primer pairs targeting nuclear ribosomal internal transcribed spacer 1 (ITS1 : forward 5′-GTCGTAACAAGGTTTCCGTA-3′ and reverse 5′-ACACGAGCCGAGTGATCC-3′), ITS2 , (forward 5′-GAACATCGACATCTTGAACG-3′, reverse 5′-GGAACGACCTGAACACCA-3′) and mitochondrial DNA, cytochrome oxidase subunit 1 (cox1 : forward 5′-GGGTTTGGAATGATTAGTC-3′ and reverse 5′-CACAGAGGCAGAAAGAACT-3′) [ – ]. .. Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template.

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: .. PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA. .. Agarose-gel purified PCR products served as templates for sequencing, with the exception that purified PCR products inserted into the promoterless pGL3-Basic vector (Promega, USA) were used for sequencing the promoter region.

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: DNA was then eluted and analyzed by quantitative real-time polymerase chain reaction (PCR) using specific primers for the ER-binding sites closest to selected ER-regulated genes. .. One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA).

Sonication:

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: Cells were fixed with 1% formaldehyde for 10 min at room temperature, lysed in RIPA (Sigma-Aldrich) buffer, sonicated and then the soluble fraction pre-cleared for 1 h with protein A/G agarose beads. .. One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA).

Binding Assay:

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9
Article Snippet: Twenty microliter of reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo). .. In vivo hAAT -integrated ROSA26 junction capture PCR was performed with a primer located within the 3′ end of hAAT (Supplementary Table , Oligo-7) and a primer binding to the 3′ ROSA26 locus, past the right homology sequence (Supplementary Table , Oligo-8).

In Vivo:

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9
Article Snippet: .. Twenty microliter of reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo). .. In vitro and in vivo EGFP -integrated junction PCR reactions also contained primers (Supplementary Table, Oligo-4 and Oligo-5) to amplify a 616 bp viral sequence to confirm the presence of viral vectors and as a loading control.

Real-time Polymerase Chain Reaction:

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: DNA was then eluted and analyzed by quantitative real-time polymerase chain reaction (PCR) using specific primers for the ER-binding sites closest to selected ER-regulated genes. .. One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA).

Isolation:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Paragraph title: RNA isolation ... The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: Total RNA was isolated using Trizol® as per manufacturer’s instructions. .. One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA).

Size-exclusion Chromatography:

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template. .. PCR was performed as follows: denaturation at 95°C for 50 sec, primer annealing at 65°C for 50 sec, and extension at 68°C for 7 min with a final extension for 5 min at 72°C.

Labeling:

Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target
Article Snippet: Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB). .. For fluorescent readouts, particles were labeled with 1/10 molar ratio Alexa Fluor 647-labeled complementary oligo.

Purification:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer. .. The products of these reactions were separately purified by electrophoresis through a 3% agarose/TAE gel and extracted from the gel using the QIAquick gel extraction kit (Qiagen).

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Using a method adapted from Smith et al. , purified ssDNA fragments were ligated to sequencing adapters with six-random-nucleotide overhangs ( ) using 50 pmol of each adaptor, 1 × T4 Ligase Buffer (Enzymatics), and 450 U of T4 DNA Ligase (Enzymatics) at 15 °C for 16 h with mixing at 800 r.p.m. (Eppendorf Thermomixer) in a volume of 25 μL per 4.5 mm square plug equivalent. .. Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min.

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: 3 μg of random hexamer (Life Technologies) was added to the fragmented, purified cRNA and incubated at 70°C for 10 min to anneal the primer. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The recognition site (R) of PspGI is CCWGG, where W = A or T. Synthetic ODNs, including a target (X ) and a probe (P ), are synthesized by Integrated DNA Technologies, Inc. and purified by HPLC.

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. Frozen cells were resuspended in purification buffer (500 mM NaCl, 50 mM Tris 8.3, 5 mM imidazole and 5 mM beta-mercaptoethanol).

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template. .. A PCR-amplified target gene fragment was purified using E.Z.N.A.

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA. .. Agarose-gel purified PCR products served as templates for sequencing, with the exception that purified PCR products inserted into the promoterless pGL3-Basic vector (Promega, USA) were used for sequencing the promoter region.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

Sequencing:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: Paragraph title: Deep Sequencing Library Preparation ... The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer.

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Paragraph title: Sequencing library preparation and analysis by end-labelling and PAGE ... Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min.

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The sequence of X is: TG T AAA CAT CCT CGA CTG GAA G , which is derived from human microRNA hsa-miR-30a.

Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes
Article Snippet: Paragraph title: Target capture sequencing using microDuMIP ... Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C.

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9
Article Snippet: Twenty microliter of reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo). .. In vitro and in vivo EGFP -integrated junction PCR reactions also contained primers (Supplementary Table, Oligo-4 and Oligo-5) to amplify a 616 bp viral sequence to confirm the presence of viral vectors and as a loading control.

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes). .. PCR amplicons were pooled and prepared for 150-bp paired-end Illumina MiSeq (Illumina, Inc., San Diego, CA, USA) sequencing using the vendor’s TruSeq library protocol.

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: Paragraph title: Sequencing of mouse Hmgcr gene for polymorphism discovery ... PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. The sequence of X is 5′-TGT AAA CAT CCT CGA CTG GAA G-3′, which is derived from human microRNA hsa-miR-30a.

Polyacrylamide Gel Electrophoresis:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Paragraph title: Sequencing library preparation and analysis by end-labelling and PAGE ... Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min.

Lysis:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: For sequencing library preparation, DNA-containing agarose plugs were phosphatase-treated as above, and phosphatase was removed by digestion in 100 μL lysis buffer per 4.5 mm square plug piece (0.4M EDTA (Invitrogen UltraPure), 2% v/v Sarkosyl (Fisher BioReagents), 0.5 mg/mL Proteinase K (New England Biolabs), pH 8) for 18 h at 50 °C, then washed five times with 1 mL TE (10 mM Tris pH 8, 1 mM EDTA) at room temperature with gentle rocking, 1 h per wash; 5′ phosphates were added by soaking each 4.5 mm plug piece in 100 μL PNK buffer (70 mM Tris, 10 mM MgCl2 , 5 mM dithiothreitol, 1 mM spermidine, pH 8) at 4 °C for 1 h, then soaking the plug piece in 70 μL reaction mix containing PNK buffer, 5 μM ATP (New England Biolabs), and 40 units of T4 polynucleotide kinase (Enzymatics; Beverly, Massachusetts, USA) at 4 °C for 2 h, then incubating it in the same reaction mix for 30 min at 37 °C. .. Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min.

IA:

Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target
Article Snippet: Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB). .. Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB).

Agarose Gel Electrophoresis:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. Libraries were size-selected between 100 and 1,000 bp to remove adaptor dimers using a 2.5% agarose gel and purified with a MinElute Gel Extraction Kit (Qiagen).

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template. .. The resulting restriction fragments were separated by electrophoresis on an ethidium bromide containing 1.5% agarose gel using 1X TAE buffer solution.

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA. .. Agarose-gel purified PCR products served as templates for sequencing, with the exception that purified PCR products inserted into the promoterless pGL3-Basic vector (Promega, USA) were used for sequencing the promoter region.

Chloramphenicol Acetyltransferase Assay:

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The sequence of X is: TG T AAA CAT CCT CGA CTG GAA G , which is derived from human microRNA hsa-miR-30a.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. The sequence of X is 5′-TGT AAA CAT CCT CGA CTG GAA G-3′, which is derived from human microRNA hsa-miR-30a.

Mouse Assay:

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: Sequencing of mouse Hmgcr gene for polymorphism discovery Genomic DNA samples of BPL/1J, BPH/2J and BPN/3J mice were obtained from the Jackson laboratory (Bar Harbor). .. PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA.

Chromatin Immunoprecipitation:

Article Title: Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses
Article Snippet: Paragraph title: Quantitative reverse transcriptase–polymerase chain reaction and chromatin immunoprecipitation ... One microgram of total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamers and 1 mM dNTPs (New England Biolabs, Beverly, MA, USA).

Plasmid Preparation:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: From each library between 108 and 109 cells were collected, and DNA was extracted using the Zymoprep Yeast Plasmid DNA Miniprep II kit (Zymo Research). .. The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer.

Article Title: Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA
Article Snippet: 2 μl of plasmid DNA solution was added to 15 μl of sample buffer and heated at 95°C for 3 min. .. Upon cooling, 15 μl of reaction buffer, 1 μl enzyme and 2 μl of 10 mM dNTPs (New England Biolabs) were added, and the reaction was incubated for 24 h at 30°C.

Article Title: Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase
Article Snippet: Reagents MuLV RT and dNTPs were purchased from New England Biolabs and Sigma, respectively. .. This expression plasmid was transformed into BL21 cells which were grown in Luria-Bertani medium until OD600 = 0.7, at which time expression was induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were supplemented with 50 µM Zinc acetate.

Article Title: Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension
Article Snippet: PCR was carried out using Phusion™ High-Fidelity DNA polymerase and dNTPs from New England Biolabs, USA. .. Agarose-gel purified PCR products served as templates for sequencing, with the exception that purified PCR products inserted into the promoterless pGL3-Basic vector (Promega, USA) were used for sequencing the promoter region.

Electrophoresis:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer. .. The products of these reactions were separately purified by electrophoresis through a 3% agarose/TAE gel and extracted from the gel using the QIAquick gel extraction kit (Qiagen).

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template. .. The resulting restriction fragments were separated by electrophoresis on an ethidium bromide containing 1.5% agarose gel using 1X TAE buffer solution.

Multiplex Assay:

Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes
Article Snippet: Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C. .. The post-amplification reaction used 1 μl of hybridized template, 10 μl of QIAGEN Multiplex PCR Master Mix (QIAGEN), 8 μl of dH2 O with 1 μl each of Capture_AmpF and Capture_AmpR primers (Supplementary Table S1).

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: Each primer was uniquely modified on the 5′end with a 10-bp multiplex identifier (MID; Roche Diagnostics Technical Bulletin TCB No.005–2009), and samples were amplified using a unique combination of forward and reverse MID-labelled primers, which allowed recovery of the amplicons per individual after demultiplexing. .. PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes).

Selection:

Article Title: Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen
Article Snippet: All 10 loci were checked for selection using LOSITAN , which suggested that one of the 10 loci (Pret-46) did not conform to expectations under neutrality. .. PCRs contained 0.2 mM dNTPs (New England Biolabs), 0.5l M forward primer, 0.5l M reverse primer,19 Phusion HF buffer, 6% DMSO,~1–3 ng genomic DNA and 0.4 U Phusion DNA Polymerase (Finnzymes).

Sample Prep:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: 1 μg of cRNA was fragmented in 1× Fragmentation Buffer (mRNA-Seq Sample Prep Kit, Illumina) at 94°C for 5 min, then placed on ice and the reaction was stopped by the addition of 20 mM EDTA. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

In Vitro:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: Each entire sample was input into the Ambion WT Expression Kit (Life Technologies) to perform double stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9
Article Snippet: .. Twenty microliter of reactions contained 0.4 µL Phire II polymerase (Thermo Fisher Scientific), 0.5 µM dNTPS (New England Biolabs), 250 µM primers, 150 ng of template DNA (in vitro) or 250 ng (in vivo). .. In vitro and in vivo EGFP -integrated junction PCR reactions also contained primers (Supplementary Table, Oligo-4 and Oligo-5) to amplify a 616 bp viral sequence to confirm the presence of viral vectors and as a loading control.

Random Hexamer Labeling:

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: 3 μg of random hexamer (Life Technologies) was added to the fragmented, purified cRNA and incubated at 70°C for 10 min to anneal the primer. .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Concentration Assay:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: .. Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. The extension reactions were purified with a QIAQUICK PCR cleanup kit (Qiagen) and concentrated to 10–20 μL per sample.

Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
Article Snippet: The cRNA was purified following the manufacturer’s instructions and the concentration was determined with a NanoDrop instrument (ThermoFisher). .. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies) with 0.625 mM dNTPs (NEB,) and 8U SUPERase RNase Inhibitor (Life Technologies) at 25°C for 10 min, then 42°C for 50 min, then 75°C for 15 min and cooled to 4°C.

Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target
Article Snippet: .. Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB). .. For fluorescent readouts, particles were labeled with 1/10 molar ratio Alexa Fluor 647-labeled complementary oligo.

DNA Purification:

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: .. Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

CTG Assay:

Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)
Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc . .. The sequence of X is: TG T AAA CAT CCT CGA CTG GAA G , which is derived from human microRNA hsa-miR-30a.

Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. The sequence of X is 5′-TGT AAA CAT CCT CGA CTG GAA G-3′, which is derived from human microRNA hsa-miR-30a.

Gel Extraction:

Article Title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning *
Article Snippet: The conditions of this reaction were 500 n m forward and reverse primers, 200 μ m dNTPs (New England Biolabs), and 3 units of Phusion High Fidelity DNA polymerase in 1× Phusion HF buffer. .. The products of these reactions were separately purified by electrophoresis through a 3% agarose/TAE gel and extracted from the gel using the QIAquick gel extraction kit (Qiagen).

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. Libraries were size-selected between 100 and 1,000 bp to remove adaptor dimers using a 2.5% agarose gel and purified with a MinElute Gel Extraction Kit (Qiagen).

Variant Assay:

Article Title: Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping
Article Snippet: Excess adapters were removed with S-300-HR spin columns (Bio-Rad), MgCl2 was added to restore magnesium concentration to 10 mM, and the 3′ adaptor was extended using 0.02 U/μL Taq-B enzyme, 1 × PCR Buffer (both, Enzymatics) and 0.2 mM (each) dNTPs (New England Biolabs) at 65 °C for 30 min. .. Libraries were amplified for 13–16 cycles (up to 21 cycles for size-selected libraries) with primers that add the full Illumina sequencing adaptor sequences (an optimized variant of TruSeq adaptors, ) and NEBNext 2 × PCR Master Mix (New England Biolabs), using an aliquot of the sample to determine the optimal number of cycles as in ref. .

Fluorescence In Situ Hybridization:

Article Title: Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan
Article Snippet: In addition, a primer pair was used as a control to detect of O. viverrini metacercariae from fish [ ]. .. Amplification of the targeted genes was carried out in a final reaction volume of 50 μl containing 1×Phusion HF buffer, 2.5 mM MgCl2 , 1 U Phusion DNA polymerase and 200 μM dNTPs (New England BioLabs Inc., Ipswich, Massachusetts, USA), 25 pmol of each primer and 20 ng DNA extracted from a single liver fluke specimen as template.

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