nuclease free water  (New England Biolabs)


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    Name:
    Nuclease free Water
    Description:
    Nuclease free Water 100 ml
    Catalog Number:
    b1500l
    Price:
    62
    Size:
    100 ml
    Category:
    Ribonuclease Protection Assays
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    Structured Review

    New England Biolabs nuclease free water
    Nuclease free Water
    Nuclease free Water 100 ml
    https://www.bioz.com/result/nuclease free water/product/New England Biolabs
    Average 95 stars, based on 69 article reviews
    Price from $9.99 to $1999.99
    nuclease free water - by Bioz Stars, 2020-02
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    Related Articles

    Clone Assay:

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: The PCR product or gBlock was digested with the appropriate restriction enzymes (NEB, Ipswich, MA), cloned into the suicide knockout plasmid pNTPS138 [ ], and transferred to the appropriate destination strain using a triparental mating strategy. .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C.

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

    Amplification:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: Briefly, library preparation was as follows: 1 μg of purified PCR amplicon was diluted to 80 μL using RT-PCR molecular grade water (Ambion). .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany
    Article Snippet: In brief, the entire 16S rRNA gene (~ 1.5kb) of selected samples was amplified using the native 16S Barcoding Kit SQK- RAB204 (Nanopore Technologies, Oxford, England). .. Each PCR reaction was performed in a total of 50 μl of reaction solution comrpising 14 μl of nuclease-free water, 10 ng of input DNA, 1 μl of barcode and 25 μl of LongAmp Taq 2X Mastermix (New England Biolabs, Frankfurt, Germany).

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: .. Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol. .. The ligation step was performed with 30 μL of DNA end-prepped, 20 μL adapter mix, and 50 μL of Blunt/TA ligase master mix (New England Biolabs, Ipswich, MA, USA).

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: PCR amplification and direct sequencing of the product verified allele substitutions. .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C.

    Article Title: The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention
    Article Snippet: .. The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA). .. Flow Cytometric Analysis SLC5A8-pLVX cells were treated for 72 h with varying concentrations of 2,4,6-THBA.

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C. .. The reactions were purified with Minelute columns and the remaining DNA was amplified to the middle of linear range by qPCR (1 µL input DNA, 25 µL reaction volume) using iTaq polymerase (Universal SYBR Green Supermix; Bio Rad) and primers listed in Supplementary Note .

    Synthesized:

    Article Title: Polycomb complexes associate with enhancers and promote oncogenic transcriptional programs in cancer through multiple mechanisms
    Article Snippet: The 10 μl qPCR reaction was set up as follows: 2 μl of transposed DNA, 0.3 μl 25 μM Ad1_noMX, 0.3 μl 25 μM Ad2.X (custom oligos synthesized by Integrated DNA Technologies, see Supplementary Data ), 5 μl NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs #M0541S), 0.1 μl 100X SYBR Green I (Thermo Fisher Scientific #S7563) and 2.3 μl nuclease-free water with the following program on a Bio-Rad CFX96 Optics Module Thermal Cycler machine: (1) 72 °C for 5 min, (2) 98 °C for 30 s, (3) 98 °C for 10 s, 63 °C for 30 s and 72 °C for 30 s, 25 cycles, (4) 72 °C for 1 min, and (5) hold at 10 °C. .. A 50 μl PCR reaction was then set up as follows: 10 μl transposed DNA, 1.5 μl 25 μM Ad1_noMX, 1.5 μl 25 μM Ad2.X (unique for each sample), 12 μl nuclease-free water, and 25 μl NEBNext High-Fidelity 2X PCR Master Mix with the same program as for the qPCR, but substituting the cycle number with the Ct-value obtained from the qPCR reaction.

    Blocking Assay:

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: The reaction was then snap-cooled on a pre-chilled freezer block. .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water.

    Real-time Polymerase Chain Reaction:

    Article Title: Polycomb complexes associate with enhancers and promote oncogenic transcriptional programs in cancer through multiple mechanisms
    Article Snippet: .. A 50 μl PCR reaction was then set up as follows: 10 μl transposed DNA, 1.5 μl 25 μM Ad1_noMX, 1.5 μl 25 μM Ad2.X (unique for each sample), 12 μl nuclease-free water, and 25 μl NEBNext High-Fidelity 2X PCR Master Mix with the same program as for the qPCR, but substituting the cycle number with the Ct-value obtained from the qPCR reaction. .. The PCR was performed on a Bio-Rad C1000 Touch Thermal Cycler.

    Article Title: The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention
    Article Snippet: .. The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA). .. Flow Cytometric Analysis SLC5A8-pLVX cells were treated for 72 h with varying concentrations of 2,4,6-THBA.

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C. .. The reactions were purified with Minelute columns and the remaining DNA was amplified to the middle of linear range by qPCR (1 µL input DNA, 25 µL reaction volume) using iTaq polymerase (Universal SYBR Green Supermix; Bio Rad) and primers listed in Supplementary Note .

    Incubation:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min. .. The ligated DNA was purified using His-tag Dynabeads.

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water. .. End-repair and dA-tailing End-repair was performed using the NEBNext End Repair Module (New England BioLabs E6050; New England BioLabs, Ipswich, MA, USA) with 6 µl of 10× end-repair buffer and 3 µl of end-repair enzyme mix added to each of the 51 µl ds cDNA samples, followed by incubation at room temperature for 25 min and clean-up using a 1.8× volume of Agencourt beads (as above), with elution in 25 µl nuclease-free water.

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: The reaction was incubated at room temperature for 15 minutes, and 1 μL HP Tether was added and incubated for an additional 10 minu tes at room temperature. .. Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol.

    Article Title: A flow cytometry-based screen for synthetic riboswitches
    Article Snippet: Using 1 ng of the double-stranded DNA pool, a 20 μl in vitro transcription reaction was prepared using the AmpliScribe™ T7-Flash™ Transcription Kit from Epicentre Biotechnologies and incubated at 37°C for 1 h. Following transcription, 1 μl of DNaseI was added to the reaction mixture to remove the DNA template. .. The RNA was resuspended in 44 μl of nuclease-free water and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C. ..

    Article Title: HLA genotyping by next-generation sequencing of complementary DNA
    Article Snippet: .. Fragmentation of biotinylated PCR products For fragmentation of the biotinylated PCR products, a 20 μl solution containing 2.4 μl of 84.2 ng/μl purified biotinylated PCR products (200 ng), 2 μl of 10× Fragmentase Reaction Buffer v2 (NEB), 13.6 μl of Nuclease-Free water, and 2 μl dsDNA fragmentase (NEB) was incubated at 37 °C for 5 min, and 7.5 μl of 0.5 M EDTA was added to inactivate the enzyme. .. Fragmented PCR products were purified using AMPure XP (recovery volume: 20 μl).

    Article Title: Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens
    Article Snippet: After purification with 1x volume AMPure XP beads (Beckman Coulter) and elution in 25 μl of nuclease-free water, dA-tailing was performed with the NEBNext dA-Tailing Module (NEB). .. For the adapter ligation step, the dA-tailed DNA was mixed with 10 μl Adapter Mix (Oxford Nanopore Technologies), 2 μl HP Adapter (Oxford Nanopore Technologies) and 50 μl Blunt/TA Ligase Master Mix (NEB) and incubated for 10 minutes at room temperature.

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C. .. The purified DNA was digested with the addition of NEBuffer 4, 1 mM adenosine 5′-triphosphate (ATP), and exonucleases I (20 units), III (100 units), and V (10 units) and incubated for 45–90 min at 37 °C.

    Activity Assay:

    Article Title: The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain
    Article Snippet: Binding assays Binding activity was evaluated by both EMSA and double-filter binding as described previously, with significant modifications to the binding buffer conditions ( , , , ). .. For EMSA, all Cdc13 proteins were assayed in 50 mM Tris–HCl, pH 7.8 at 4°C, 75 mM KCl, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin and 15% glycerol. ssDNA oligonucleotides (Integrated DNA Technologies) were resuspended in nuclease-free water and 5′-end-labeled using γ-32 P-ATP with T4 polynucleotide kinase (New England Biolabs).

    Expressing:

    Article Title: A flow cytometry-based screen for synthetic riboswitches
    Article Snippet: Structure probing Synthetic oligonucleotides SAL-073 and its reverse complement, SAL-074 were mixed, heated to 95°C for 2 h and cooled 1°C per minute to 4°C to yield a double-stranded DNA pool with a T7 RNA polymerase promoter fused directly 5′ to the mTCT4-8 aptamer and expression platform of switch SAL-12.1. .. The RNA was resuspended in 44 μl of nuclease-free water and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C. .. Strains expressing xylose-inducible CFP, GFP, YFP, or mCherry from a plasmid integrated at the xylX locus.

    Flow Cytometry:

    Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8+ T Cells on a Single-Cell Level
    Article Snippet: .. Single cells were flow-sorted into 96-well plates containing 2 μL of nuclease-free water with 0.2% Triton X-100 and 4 U murine RNase inhibitor (NEB), centrifuged, and frozen at −80°C. .. After thawing, 2 μL of the primer mix [5 mM dNTP (Invitrogen), 0.5 μM oligo-dT primer, 4 U murine RNase inhibitor] was added to each well.

    Ligation:

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol. .. The ligation step was performed with 30 μL of DNA end-prepped, 20 μL adapter mix, and 50 μL of Blunt/TA ligase master mix (New England Biolabs, Ipswich, MA, USA).

    Article Title: Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions
    Article Snippet: Gap filling and ligation was performed for 30 min at 65 °C. .. Linear DNA Digestion Solution (volume of 48 μl) was composed of 24μl of nuclease free water, 6 μl of Exonuclease I (20 units/μl), 6 μl of Exonuclease III (100 units/μl), 6 μl of Lambda Exonuclease (5 units/μl) and 6 μl Exonuclease VII (10 units/μl) (all from NEB).

    Article Title: Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens
    Article Snippet: After purification with 1x volume AMPure XP beads (Beckman Coulter) and elution in 25 μl of nuclease-free water, dA-tailing was performed with the NEBNext dA-Tailing Module (NEB). .. For the adapter ligation step, the dA-tailed DNA was mixed with 10 μl Adapter Mix (Oxford Nanopore Technologies), 2 μl HP Adapter (Oxford Nanopore Technologies) and 50 μl Blunt/TA Ligase Master Mix (NEB) and incubated for 10 minutes at room temperature.

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The End-repaired step was followed by a dA-tailing step of dsDNA product using the NEBNext dA-Tailing Module (New England Biolabs), prior to ligation of ONT-specific adapters with Blunt/TA Ligase Master Mix (New England Biolabs).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: The end repaired DNA was then purified using 100 μL Agencourt AMPure XP beads (Beckman Coulter Inc., Pasadena, CA, USA), as per the manufacturer’s instructions with elution into 25 μL of RT-PCR molecular grade water (Ambion). .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention
    Article Snippet: The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA). .. The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA).

    DNA Sequencing:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: The Oxford Nanopore MinION Genomic DNA Sequencing kit (version 4) was used to prepare purified PCR amplicons (as for the Illumina MiSeq; see above) for sequencing according to the manufacturer’s instructions. .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: The adapter-ligated amplicon was recovered using MyOne C1-beads (Life Technologies, Carlsbad, CA, USA) and rinsed with washing buffer provided by the Genomic DNA Sequencing Kit SQK-MAP006 (Oxford Nanopore Technologies, Oxford, UK). .. Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol.

    Article Title: Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens
    Article Snippet: MinION library preparation was performed using components from the Genomic DNA Sequencing Kit (Oxford Nanopore Technologies) as follows. .. After purification with 1x volume AMPure XP beads (Beckman Coulter) and elution in 25 μl of nuclease-free water, dA-tailing was performed with the NEBNext dA-Tailing Module (NEB).

    Polymerase Chain Reaction:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: Briefly, library preparation was as follows: 1 μg of purified PCR amplicon was diluted to 80 μL using RT-PCR molecular grade water (Ambion). .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany
    Article Snippet: .. Each PCR reaction was performed in a total of 50 μl of reaction solution comrpising 14 μl of nuclease-free water, 10 ng of input DNA, 1 μl of barcode and 25 μl of LongAmp Taq 2X Mastermix (New England Biolabs, Frankfurt, Germany). .. The following PCR protocol was performed: 95 °C for 1 min; 25 cycles at 95 °C for 20 s, 55 °C for 30 s and 65 °C for 2 min, and a final extension step at 72 °C for 5 min. PCR products were purified using 30 μl of AMPure XP DNA beads (Beckman Coulter).

    Article Title: Polycomb complexes associate with enhancers and promote oncogenic transcriptional programs in cancer through multiple mechanisms
    Article Snippet: .. A 50 μl PCR reaction was then set up as follows: 10 μl transposed DNA, 1.5 μl 25 μM Ad1_noMX, 1.5 μl 25 μM Ad2.X (unique for each sample), 12 μl nuclease-free water, and 25 μl NEBNext High-Fidelity 2X PCR Master Mix with the same program as for the qPCR, but substituting the cycle number with the Ct-value obtained from the qPCR reaction. .. The PCR was performed on a Bio-Rad C1000 Touch Thermal Cycler.

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C. ..

    Article Title: HLA genotyping by next-generation sequencing of complementary DNA
    Article Snippet: .. Fragmentation of biotinylated PCR products For fragmentation of the biotinylated PCR products, a 20 μl solution containing 2.4 μl of 84.2 ng/μl purified biotinylated PCR products (200 ng), 2 μl of 10× Fragmentase Reaction Buffer v2 (NEB), 13.6 μl of Nuclease-Free water, and 2 μl dsDNA fragmentase (NEB) was incubated at 37 °C for 5 min, and 7.5 μl of 0.5 M EDTA was added to inactivate the enzyme. .. Fragmented PCR products were purified using AMPure XP (recovery volume: 20 μl).

    Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8+ T Cells on a Single-Cell Level
    Article Snippet: Single cells were flow-sorted into 96-well plates containing 2 μL of nuclease-free water with 0.2% Triton X-100 and 4 U murine RNase inhibitor (NEB), centrifuged, and frozen at −80°C. .. The number of pre-amplification PCR cycles was increased to 22 to ensure there was sufficient cDNA for downstream analysis.

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

    Binding Assay:

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water. .. End-repair and dA-tailing End-repair was performed using the NEBNext End Repair Module (New England BioLabs E6050; New England BioLabs, Ipswich, MA, USA) with 6 µl of 10× end-repair buffer and 3 µl of end-repair enzyme mix added to each of the 51 µl ds cDNA samples, followed by incubation at room temperature for 25 min and clean-up using a 1.8× volume of Agencourt beads (as above), with elution in 25 µl nuclease-free water.

    Article Title: The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain
    Article Snippet: Paragraph title: Binding assays ... For EMSA, all Cdc13 proteins were assayed in 50 mM Tris–HCl, pH 7.8 at 4°C, 75 mM KCl, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin and 15% glycerol. ssDNA oligonucleotides (Integrated DNA Technologies) were resuspended in nuclease-free water and 5′-end-labeled using γ-32 P-ATP with T4 polynucleotide kinase (New England Biolabs).

    Nucleic Acid Electrophoresis:

    Article Title: A flow cytometry-based screen for synthetic riboswitches
    Article Snippet: The RNA was resuspended in 44 μl of nuclease-free water and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs). .. Radiolabeled RNA was purified using denaturing gel electrophoresis.

    RNA Sequencing Assay:

    Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8+ T Cells on a Single-Cell Level
    Article Snippet: Paragraph title: Single-Cell RNA Sequencing ... Single cells were flow-sorted into 96-well plates containing 2 μL of nuclease-free water with 0.2% Triton X-100 and 4 U murine RNase inhibitor (NEB), centrifuged, and frozen at −80°C.

    RNA HS Assay:

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: mRNA extraction and double-stranded cDNA synthesis Total RNA was extracted from the venom glands of two Echis coloratus (snap-frozen after removal and stored at −80 °C ( ; )) using TriReagent (Sigma T9424; Sigma Aldrich, St. Louis, MO, USA) and mRNA purified using the polyA Spin mRNA Isolation Kit (New England BioLabs S1560; New England BioLabs, Ipswich, MA, USA). mRNA was quantified using a Qubit fluorometer (Qubit RNA HS Assay Kit Q32852; Thermo Scientific, Waltham, MA, USA) and reverse transcription carried out using 120 ng (Eco6) or 240 ng of mRNA (Eco8). .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water.

    Isolation:

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: mRNA extraction and double-stranded cDNA synthesis Total RNA was extracted from the venom glands of two Echis coloratus (snap-frozen after removal and stored at −80 °C ( ; )) using TriReagent (Sigma T9424; Sigma Aldrich, St. Louis, MO, USA) and mRNA purified using the polyA Spin mRNA Isolation Kit (New England BioLabs S1560; New England BioLabs, Ipswich, MA, USA). mRNA was quantified using a Qubit fluorometer (Qubit RNA HS Assay Kit Q32852; Thermo Scientific, Waltham, MA, USA) and reverse transcription carried out using 120 ng (Eco6) or 240 ng of mRNA (Eco8). .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water.

    Article Title: The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention
    Article Snippet: Paragraph title: 4.8. RNA Isolation and qRT-PCR ... The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA).

    Labeling:

    Article Title: The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain
    Article Snippet: For EMSA, all Cdc13 proteins were assayed in 50 mM Tris–HCl, pH 7.8 at 4°C, 75 mM KCl, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin and 15% glycerol. ssDNA oligonucleotides (Integrated DNA Technologies) were resuspended in nuclease-free water and 5′-end-labeled using γ-32 P-ATP with T4 polynucleotide kinase (New England Biolabs). .. Free ATP was removed with G25 spin columns (GE Healthcare), and labeled ssDNA ligands were diluted to 2× final concentration in binding buffer.

    Purification:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: The end repaired DNA was then purified using 100 μL Agencourt AMPure XP beads (Beckman Coulter Inc., Pasadena, CA, USA), as per the manufacturer’s instructions with elution into 25 μL of RT-PCR molecular grade water (Ambion). .. 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany
    Article Snippet: Each PCR reaction was performed in a total of 50 μl of reaction solution comrpising 14 μl of nuclease-free water, 10 ng of input DNA, 1 μl of barcode and 25 μl of LongAmp Taq 2X Mastermix (New England Biolabs, Frankfurt, Germany). .. The following PCR protocol was performed: 95 °C for 1 min; 25 cycles at 95 °C for 20 s, 55 °C for 30 s and 65 °C for 2 min, and a final extension step at 72 °C for 5 min. PCR products were purified using 30 μl of AMPure XP DNA beads (Beckman Coulter).

    Article Title: Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing
    Article Snippet: .. Second strand synthesis was performed with the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs E6111; New England BioLabs, Ipswich, MA, USA), using 45 µl of nuclease-free water, 10 µl of NEBNext Second Strand Synthesis Reaction Buffer and 5 µl of NEBNext Second Strand Synthesis Enzyme Mix, with incubation at 16 °C for 1 h. Double-stranded cDNA (ds cDNA) was purified using a 1.8× volume of Agencourt AMPure XP beads (Beckman Coulter A63880), with a 5 min binding step (with gentle shaking), two washes in 200 µl 70% ethanol and elution in 51 µl nuclease-free water. .. End-repair and dA-tailing End-repair was performed using the NEBNext End Repair Module (New England BioLabs E6050; New England BioLabs, Ipswich, MA, USA) with 6 µl of 10× end-repair buffer and 3 µl of end-repair enzyme mix added to each of the 51 µl ds cDNA samples, followed by incubation at room temperature for 25 min and clean-up using a 1.8× volume of Agencourt beads (as above), with elution in 25 µl nuclease-free water.

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: .. Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol. .. The ligation step was performed with 30 μL of DNA end-prepped, 20 μL adapter mix, and 50 μL of Blunt/TA ligase master mix (New England Biolabs, Ipswich, MA, USA).

    Article Title: A flow cytometry-based screen for synthetic riboswitches
    Article Snippet: The RNA was resuspended in 44 μl of nuclease-free water and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs). .. Radiolabeled RNA was purified using denaturing gel electrophoresis.

    Article Title: HLA genotyping by next-generation sequencing of complementary DNA
    Article Snippet: .. Fragmentation of biotinylated PCR products For fragmentation of the biotinylated PCR products, a 20 μl solution containing 2.4 μl of 84.2 ng/μl purified biotinylated PCR products (200 ng), 2 μl of 10× Fragmentase Reaction Buffer v2 (NEB), 13.6 μl of Nuclease-Free water, and 2 μl dsDNA fragmentase (NEB) was incubated at 37 °C for 5 min, and 7.5 μl of 0.5 M EDTA was added to inactivate the enzyme. .. Fragmented PCR products were purified using AMPure XP (recovery volume: 20 μl).

    Article Title: Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions
    Article Snippet: After digestion, the capture reaction was purified using AMPure beads (1.8 X and washed with 70% ethanol) and eluted in 25 μl of DNAse free water. .. Linear DNA Digestion Solution (volume of 48 μl) was composed of 24μl of nuclease free water, 6 μl of Exonuclease I (20 units/μl), 6 μl of Exonuclease III (100 units/μl), 6 μl of Lambda Exonuclease (5 units/μl) and 6 μl Exonuclease VII (10 units/μl) (all from NEB).

    Article Title: Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens
    Article Snippet: .. After purification with 1x volume AMPure XP beads (Beckman Coulter) and elution in 25 μl of nuclease-free water, dA-tailing was performed with the NEBNext dA-Tailing Module (NEB). .. For the adapter ligation step, the dA-tailed DNA was mixed with 10 μl Adapter Mix (Oxford Nanopore Technologies), 2 μl HP Adapter (Oxford Nanopore Technologies) and 50 μl Blunt/TA Ligase Master Mix (NEB) and incubated for 10 minutes at room temperature.

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: MinION™ sequencing libraries were prepared directly from the total products of HBV full-length genome PCR reactions after purification with the MinElute PCR Purification kit (Qiagen) and from cloned full-length HBV genomes after enzymatic linearization of the plasmid vector; linearized plasmids were purified with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France). .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France).

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C. .. Addition of PB buffer (Qiagen, 200 µL) stopped the reaction, and DNA was purified with Minelute columns (Qiagen).

    Sequencing:

    Article Title: MinION nanopore sequencing of an influenza genome
    Article Snippet: Paragraph title: MinION Library Preparation and Sequencing ... 8 μL nuclease-free water was added to 30 μL of the dA-tailed DNA, and then 10 μL adapter mix, 2 μL HP adapter and 50 μL Blunt/TA DNA ligase mastermix (New England Biolabs) were added in this order, and then incubated at 25°C for 10 min.

    Article Title: Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany
    Article Snippet: Paragraph title: 16S full length rRNA gene sequencing using Nanopore ... Each PCR reaction was performed in a total of 50 μl of reaction solution comrpising 14 μl of nuclease-free water, 10 ng of input DNA, 1 μl of barcode and 25 μl of LongAmp Taq 2X Mastermix (New England Biolabs, Frankfurt, Germany).

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: The R9.4 sequencing library was obtained by processing 600 ng of purified amplicon DNA (0.15 per mock community plus 0.15 μg of 2 extra query samples consisting of amplicons obtained from a genomic DNA mix of several uncharacterized microbial isolates) with SQK-LSK108 sequencing (Oxford Nanopore Technologies, Oxford, UK) for 1D reads, following the manufacturer's instructions. .. Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol.

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C. ..

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: MinION™ sequencing libraries were prepared directly from the total products of HBV full-length genome PCR reactions after purification with the MinElute PCR Purification kit (Qiagen) and from cloned full-length HBV genomes after enzymatic linearization of the plasmid vector; linearized plasmids were purified with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France). .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France).

    Quantitative RT-PCR:

    Article Title: The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention
    Article Snippet: Paragraph title: 4.8. RNA Isolation and qRT-PCR ... The final volume was adjusted to 20 µL using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50 °C for 2 min, 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 min; and one melt cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse: ACCCTGTTGCTGT AGCCA).

    Plasmid Preparation:

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: The PCR product or gBlock was digested with the appropriate restriction enzymes (NEB, Ipswich, MA), cloned into the suicide knockout plasmid pNTPS138 [ ], and transferred to the appropriate destination strain using a triparental mating strategy. .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C.

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: MinION™ sequencing libraries were prepared directly from the total products of HBV full-length genome PCR reactions after purification with the MinElute PCR Purification kit (Qiagen) and from cloned full-length HBV genomes after enzymatic linearization of the plasmid vector; linearized plasmids were purified with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France). .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France).

    SYBR Green Assay:

    Article Title: Polycomb complexes associate with enhancers and promote oncogenic transcriptional programs in cancer through multiple mechanisms
    Article Snippet: The 10 μl qPCR reaction was set up as follows: 2 μl of transposed DNA, 0.3 μl 25 μM Ad1_noMX, 0.3 μl 25 μM Ad2.X (custom oligos synthesized by Integrated DNA Technologies, see Supplementary Data ), 5 μl NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs #M0541S), 0.1 μl 100X SYBR Green I (Thermo Fisher Scientific #S7563) and 2.3 μl nuclease-free water with the following program on a Bio-Rad CFX96 Optics Module Thermal Cycler machine: (1) 72 °C for 5 min, (2) 98 °C for 30 s, (3) 98 °C for 10 s, 63 °C for 30 s and 72 °C for 30 s, 25 cycles, (4) 72 °C for 1 min, and (5) hold at 10 °C. .. A 50 μl PCR reaction was then set up as follows: 10 μl transposed DNA, 1.5 μl 25 μM Ad1_noMX, 1.5 μl 25 μM Ad2.X (unique for each sample), 12 μl nuclease-free water, and 25 μl NEBNext High-Fidelity 2X PCR Master Mix with the same program as for the qPCR, but substituting the cycle number with the Ct-value obtained from the qPCR reaction.

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C. .. The reactions were purified with Minelute columns and the remaining DNA was amplified to the middle of linear range by qPCR (1 µL input DNA, 25 µL reaction volume) using iTaq polymerase (Universal SYBR Green Supermix; Bio Rad) and primers listed in Supplementary Note .

    Multiplex Assay:

    Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
    Article Snippet: Briefly, the 600 ng of amplicon DNA diluted in 50 μL nuclease-free water were processed for end repair using the NEBNextUltra II End Repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and washing twice with 200 μL of fresh 70% ethanol. .. Samples for the Illumina MiSeq approach were sent to the National Center for Genomic Analaysis (CNAG, Barcelona, Spain) for multiplex sequencing in 1 lane of MiSeq instrument with 2 × 300 paired-end configuration.

    In Vitro:

    Article Title: A flow cytometry-based screen for synthetic riboswitches
    Article Snippet: Using 1 ng of the double-stranded DNA pool, a 20 μl in vitro transcription reaction was prepared using the AmpliScribe™ T7-Flash™ Transcription Kit from Epicentre Biotechnologies and incubated at 37°C for 1 h. Following transcription, 1 μl of DNaseI was added to the reaction mixture to remove the DNA template. .. The RNA was resuspended in 44 μl of nuclease-free water and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
    Article Snippet: Paragraph title: In vitro recombination assays ... In a total reaction volume of 50 µL, recombinase (0.66 pmol for a 1:3 ratio of protein:DNA) was mixed with 1 pmol per oligonucleotide in nuclease-free water and Cre Recombinase Buffer (NEB) for 30 min at 37 °C.

    Knock-Out:

    Article Title: Exopolysaccharide production in Caulobacter crescentus: A resource allocation trade-off between protection and proliferation
    Article Snippet: The PCR product or gBlock was digested with the appropriate restriction enzymes (NEB, Ipswich, MA), cloned into the suicide knockout plasmid pNTPS138 [ ], and transferred to the appropriate destination strain using a triparental mating strategy. .. When used as a template for Sanger sequencing, 10 μl of the fresh PCR product was mixed with 8.5 μL nuclease free water, 1.0 μl NEB Buffer 4, 0.1 μL ExoT (NEB, Ipswich, MA) and 0.25 μL rSAP (NEB, Ipswich, MA) incubated at 37°C for 30 min and at 80°C for 15 min, and stored at -20°C.

    Concentration Assay:

    Article Title: The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain
    Article Snippet: For EMSA, all Cdc13 proteins were assayed in 50 mM Tris–HCl, pH 7.8 at 4°C, 75 mM KCl, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin and 15% glycerol. ssDNA oligonucleotides (Integrated DNA Technologies) were resuspended in nuclease-free water and 5′-end-labeled using γ-32 P-ATP with T4 polynucleotide kinase (New England Biolabs). .. Free ATP was removed with G25 spin columns (GE Healthcare), and labeled ssDNA ligands were diluted to 2× final concentration in binding buffer.

    Nanopore Sequencing:

    Article Title: Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
    Article Snippet: .. Briefly, the protocol for preparing a nanopore sequencing with SQK-MAP004 is as follows: 500 ng to 1μg of PCR products or cloned HBV DNA were diluted to 80μL using nuclease-free water and end-repaired using the NEBNext End Repair Module (New England Biolabs, Evry, France). .. The end-repaired products were then purified using 1.0 x volume of AMPure XP beads (Beckman-Coulter) and eluted in 25μL of nuclease-free.

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