buffer 2  (New England Biolabs)


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    Structured Review

    New England Biolabs buffer 2
    Buffer 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/buffer 2/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    buffer 2 - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: For blots that detected mmu-miR-125b ligations used the 17-mer Universal miRNA cloning linker (New England Biolabs Inc.) because the pre-adenylated DNA adapter used for the qPCR experiments is the same size as this miRNA and obscured hybridization. .. Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.).

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: PCR products were cloned via LIC into pL2, a binary vector based on pGreenII (Hellens et al ., ), containing an nptII ). .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells.

    Amplification:

    Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum
    Article Snippet: Paragraph title: Amplified fragments length polymorphism (AFLP) analysis ... The restriction reaction contained 2 units of Eco RI, 2 units of Mse I, 1.25 μL of buffer 2 (NEB), 100 ng·μL−1 BSA, and 250 ng of DNA in a final volume of 12.5 μL and was incubated for 3 h at 37°C.

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. The tagged cDNA was purified with DNA Clean & Concentrator columns (Zymo) and amplified using the Failsafe PCR system (Premix E, Epicentre) using the following forward primer: 5΄-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3΄, and one of the following reverse primers (Illumina, indexed sequence is shown in bold font): 5΄-CAAGCAGAAGACGGCATACGAGAT CGTGAT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT ACATCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT GCCTAA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT TGGTCA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT CACTGT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, or 5΄-CAAGCAGAAGACGGCATACGAGAT ATTGGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄.

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: 150 ng of genomic DNA was then used for PCR amplification. .. PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×.

    Synthesized:

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements
    Article Snippet: For random 8mer libraries, an oligonucleotide containing a Xho I site, a random 8 nucleotide sequence, a Bam HI site, a hairpin sequence, and a Bam HI site was synthesized by IDT (Additional file ). .. The oligonucleotide was annealed by heating to 95 °C for 5 min in the presence of Buffer 2 (NEB) and 0.2 mM dNTPs, then cooled on ice for 3 min, thus creating a partially double stranded structure and internally primed substrate for DNA polymerase.

    Article Title: Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA
    Article Snippet: .. The E. coli tRNAMet DNA template was synthesized in a 2-ml reaction containing 4 μM EctRNAMet 5 and EctRNAMet 3 primers, 400 μM deoxynucleoside triphosphates, 50 U of Klenow exo− DNA polymerase, and 1× buffer 2 from New England BioLabs. .. The reaction mixture was incubated for 20 cycles using a Thermocycler at 10°C for 10 s and 37°C for 30 s. Transcription reaction (8 ml) contained 2 ml of DNA template, 5 mM each nucleoside triphosphates, 20 mM spermidine, 40 mM dithiothreitol (DTT), 250 mM HEPES (pH 7.5), 30 mM MgCl2 , 20 μg of bovine serum albumin, 40 μg of T7 RNA polymerase/ml, and 60 U of RNase inhibitor.

    Construct:

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: SRD5A2 promoter-luciferase constructs (1 μg) were methylated in vitro by M.SssI (New England Biolabs, Ipswich, MA) as recommended by the manufacturer. .. Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours.

    Real-time Polymerase Chain Reaction:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: For blots that detected mmu-miR-125b ligations used the 17-mer Universal miRNA cloning linker (New England Biolabs Inc.) because the pre-adenylated DNA adapter used for the qPCR experiments is the same size as this miRNA and obscured hybridization. .. Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.).

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. Library size distribution was analyzed by D1000 ScreenTape analysis (Agilent) and then quantitated by qPCR.

    Microarray:

    Article Title: Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis
    Article Snippet: Each DNA sample was sheared by passing it through a 30-gauge needle 65 times, and a 4-μg portion of each sheared DNA sample was added to a PCR tube containing 3 μg of M. catarrhalis genome-directed primers ( ) in 20 μl of buffer 2 (New England BioLabs, Ipswich, MA). .. Each of the Cy3-labeled DNA samples was cleaned as described below and hybridized to individual DNA microarray slides as previously described ( ).

    Incubation:

    Article Title: Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders
    Article Snippet: To the remaining 45 μl PCR mix, 5 μl of Buffer 2 (NEB) was added and the reactions were heated at 95°C for 10 minutes in a PCR machine and cooled slowly to 25°C to produce heteroduplexes. .. All reactions were incubated at 37°C for 30 minutes, followed by addition of 10 μl of 6X loading dye and analysis on an agarose/TAE gel.

    Article Title: Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis
    Article Snippet: Each DNA sample was sheared by passing it through a 30-gauge needle 65 times, and a 4-μg portion of each sheared DNA sample was added to a PCR tube containing 3 μg of M. catarrhalis genome-directed primers ( ) in 20 μl of buffer 2 (New England BioLabs, Ipswich, MA). .. The final reaction volume was brought to 49 μl with water and allowed to incubate at room temperature for 2 min. Klenow enzyme (1 μl) (NEB) was added and incubated at room temperature for 5 min. Each reaction tube was placed at 37°C for 2.5 h, and then an additional 1 μl of Klenow enzyme was added before undergoing a 2-h incubation at 37°C.

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: .. Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours. .. M.SssI was heat-inactivated at 65°C for 20 minutes, and removed by a PCR purification kit (Qiagen).

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: .. After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Size selection following the ligation and PCR steps was performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads respectively (Beckman Coulter - A63880).

    Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum
    Article Snippet: .. The restriction reaction contained 2 units of Eco RI, 2 units of Mse I, 1.25 μL of buffer 2 (NEB), 100 ng·μL−1 BSA, and 250 ng of DNA in a final volume of 12.5 μL and was incubated for 3 h at 37°C. .. The ligation reaction contained 5 pmol of Eco RI adaptors, 50 pmol of Mse I adaptors, 1.25 μL T4 DNA ligase buffer (Promega), 0.75 units of T4 DNA ligase (Promega, Madison, WI), and 6.25 μL of the digested products in a final volume of 12.5 μL.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer
    Article Snippet: .. Quantitation of nucleosides by HPLC Genomic DNA was extracted with Gene Jet Genomic DNA extraction Kit (Thermo Fisher) or TRI Reagent (Sigma Aldrich), incubated with RNase A/T1 (Thermo Fisher) in buffer 2 (NEB), phenol/chloroform extracted and precipitated with ethanol. ..

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×. .. Heteroduplexes were incubated for 30 min at 37 °C in presence of 10 units of T7 Endonuclease I (NEB).

    Article Title: A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish
    Article Snippet: 150 ng) were denatured and re-annealed in a thermocycler by incubation for 10 min at 95°C, 95°C–85°C at a rate of -2°C/s, 85°C–25°C at a rate of -0.3°C/s and then held at 4°C. .. The re-annealed DNAs were then treated with 5 U (0.5 μl) of T7 endonuclease I (New England Biolabs) upon addition of 1.5 μl buffer 2 (New England Biolabs) and 3.5 μl H2 O to a final volume of 15 μl.

    Article Title: Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA
    Article Snippet: The E. coli tRNAMet DNA template was synthesized in a 2-ml reaction containing 4 μM EctRNAMet 5 and EctRNAMet 3 primers, 400 μM deoxynucleoside triphosphates, 50 U of Klenow exo− DNA polymerase, and 1× buffer 2 from New England BioLabs. .. The reaction mixture was incubated for 20 cycles using a Thermocycler at 10°C for 10 s and 37°C for 30 s. Transcription reaction (8 ml) contained 2 ml of DNA template, 5 mM each nucleoside triphosphates, 20 mM spermidine, 40 mM dithiothreitol (DTT), 250 mM HEPES (pH 7.5), 30 mM MgCl2 , 20 μg of bovine serum albumin, 40 μg of T7 RNA polymerase/ml, and 60 U of RNase inhibitor.

    Transformation Assay:

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Hybridization:

    Article Title: Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis
    Article Snippet: Paragraph title: DNA:DNA hybridization. ... Each DNA sample was sheared by passing it through a 30-gauge needle 65 times, and a 4-μg portion of each sheared DNA sample was added to a PCR tube containing 3 μg of M. catarrhalis genome-directed primers ( ) in 20 μl of buffer 2 (New England BioLabs, Ipswich, MA).

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: For blots that detected mmu-miR-125b ligations used the 17-mer Universal miRNA cloning linker (New England Biolabs Inc.) because the pre-adenylated DNA adapter used for the qPCR experiments is the same size as this miRNA and obscured hybridization. .. Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.).

    High Performance Liquid Chromatography:

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer
    Article Snippet: .. Quantitation of nucleosides by HPLC Genomic DNA was extracted with Gene Jet Genomic DNA extraction Kit (Thermo Fisher) or TRI Reagent (Sigma Aldrich), incubated with RNase A/T1 (Thermo Fisher) in buffer 2 (NEB), phenol/chloroform extracted and precipitated with ethanol. ..

    Electroporation:

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Binary vectors were extracted from E. coli and transformed together with pSoup, required for the replication of pGreenII‐based vectors in Agrobacterium (Hellens et al ., ), into Agrobacterium tumefaciens GV3101 (pMP90) by electroporation.

    Flow Cytometry:

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer
    Article Snippet: Quantitation of nucleosides by HPLC Genomic DNA was extracted with Gene Jet Genomic DNA extraction Kit (Thermo Fisher) or TRI Reagent (Sigma Aldrich), incubated with RNase A/T1 (Thermo Fisher) in buffer 2 (NEB), phenol/chloroform extracted and precipitated with ethanol. .. Buffer A was 100 mM ammonium acetate pH 6.5, buffer B was 40% acetonitrile, and the flow rate 0.4 ml/min.

    Ligation:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: .. After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Size selection following the ligation and PCR steps was performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads respectively (Beckman Coulter - A63880).

    Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum
    Article Snippet: The restriction reaction contained 2 units of Eco RI, 2 units of Mse I, 1.25 μL of buffer 2 (NEB), 100 ng·μL−1 BSA, and 250 ng of DNA in a final volume of 12.5 μL and was incubated for 3 h at 37°C. .. The ligation reaction contained 5 pmol of Eco RI adaptors, 50 pmol of Mse I adaptors, 1.25 μL T4 DNA ligase buffer (Promega), 0.75 units of T4 DNA ligase (Promega, Madison, WI), and 6.25 μL of the digested products in a final volume of 12.5 μL.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: The I‐7 ), designed for ligation‐independent cloning (LIC), and iProof high‐fidelity DNA polymerase (BioRad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells.

    Methylation:

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: Paragraph title: In Vitro Methylation ... Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours.

    Northern Blot:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Paragraph title: Northern blots ... Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.).

    Generated:

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements
    Article Snippet: Insert generation for 8mers For individual 8mers, two oligonucleotides (IDT) were synthesized so that when they were annealed, they generated termini corresponding to sites digested by Xho I (5′ terminus) and Bam HI (3′ terminus) of the 8mer (Additional file ). .. The oligonucleotide was annealed by heating to 95 °C for 5 min in the presence of Buffer 2 (NEB) and 0.2 mM dNTPs, then cooled on ice for 3 min, thus creating a partially double stranded structure and internally primed substrate for DNA polymerase.

    DNA Sequencing:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Replicate 1 of the H3K122ac and H3K64ac ChIPs was sequenced at The Danish National High-Throughput DNA sequencing Center (Copenhagen; 42 base single end reads).

    Sequencing:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: For the detection of all of the piRNAs, the 21-mer pre-adenylated DNA adapter (sequence above) was used. .. Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.).

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: Paragraph title: ChIP-seq library preparation and Deep Sequencing ... After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. The tagged cDNA was purified with DNA Clean & Concentrator columns (Zymo) and amplified using the Failsafe PCR system (Premix E, Epicentre) using the following forward primer: 5΄-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3΄, and one of the following reverse primers (Illumina, indexed sequence is shown in bold font): 5΄-CAAGCAGAAGACGGCATACGAGAT CGTGAT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT ACATCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT GCCTAA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT TGGTCA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT CACTGT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, or 5΄-CAAGCAGAAGACGGCATACGAGAT ATTGGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄.

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements
    Article Snippet: For random 8mer libraries, an oligonucleotide containing a Xho I site, a random 8 nucleotide sequence, a Bam HI site, a hairpin sequence, and a Bam HI site was synthesized by IDT (Additional file ). .. The oligonucleotide was annealed by heating to 95 °C for 5 min in the presence of Buffer 2 (NEB) and 0.2 mM dNTPs, then cooled on ice for 3 min, thus creating a partially double stranded structure and internally primed substrate for DNA polymerase.

    ChIP-sequencing:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: Paragraph title: ChIP-seq library preparation and Deep Sequencing ... After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight.

    DNA Extraction:

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer
    Article Snippet: .. Quantitation of nucleosides by HPLC Genomic DNA was extracted with Gene Jet Genomic DNA extraction Kit (Thermo Fisher) or TRI Reagent (Sigma Aldrich), incubated with RNase A/T1 (Thermo Fisher) in buffer 2 (NEB), phenol/chloroform extracted and precipitated with ethanol. ..

    Magnetic Beads:

    Article Title: RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation
    Article Snippet: After heatinactivation (80 o C for 15 min), NbBC IA (5U) was added during 1 h. On ice, streptavidin-coated magnetic beads (100 ng) were used to pull down the biotinylated primer PCR2a. .. After the supernatant was replaced by 100 μl buffer 2 (NEB) containing primer 3 (100 nM), and dNTPs (10 mM each) the sample was heated (70 o C for 20 min) to inactivate the enzyme and release the nicked hairpins from the beads.

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. Reactions were heated at 95°C for 1 min, followed by 18 cycles of 95°C for 30 s, 55°C for 30 s and 68°C for 3 min with a final extension step at 68°C for 7 min. PCR products were purified using Agencourt AMPure XP magnetic beads at a 1:1 volume ratio.

    Isolation:

    Article Title: RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation
    Article Snippet: After the supernatant was replaced by 100 μl buffer 2 (NEB) containing primer 3 (100 nM), and dNTPs (10 mM each) the sample was heated (70 o C for 20 min) to inactivate the enzyme and release the nicked hairpins from the beads. .. The fragments were separated (4.5% agarose gel) and 91 bp fragments were isolated from the gel by freeze squeezing and phenol extraction.

    Article Title: Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis
    Article Snippet: Genomic DNA was prepared from M. catarrhalis ATCC 43617 and O35EΔ mapA with the Easy-DNA isolation kit. .. Each DNA sample was sheared by passing it through a 30-gauge needle 65 times, and a 4-μg portion of each sheared DNA sample was added to a PCR tube containing 3 μg of M. catarrhalis genome-directed primers ( ) in 20 μl of buffer 2 (New England BioLabs, Ipswich, MA).

    Article Title: Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA
    Article Snippet: Paragraph title: Synthesis and isolation of synthetic tRNA. ... The E. coli tRNAMet DNA template was synthesized in a 2-ml reaction containing 4 μM EctRNAMet 5 and EctRNAMet 3 primers, 400 μM deoxynucleoside triphosphates, 50 U of Klenow exo− DNA polymerase, and 1× buffer 2 from New England BioLabs.

    Labeling:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: .. Labeling reactions contained 120 μCi of [α-32 P]dATP, 5 U of Klenow Fragment (3′ → 5′ exo-), in 1× buffer 2 (New England Biolabs Inc.). .. Unincorporated nucleotides were removed using G-25 microspin columns (GE Healthcare).

    Purification:

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours. .. M.SssI was heat-inactivated at 65°C for 20 minutes, and removed by a PCR purification kit (Qiagen).

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: ChIP-seq library preparation and Deep Sequencing Libraries were prepared as previously described with the following modifications: No purification was performed between the A-tailing and ligation reactions. .. After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: .. RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. The tagged cDNA was purified with DNA Clean & Concentrator columns (Zymo) and amplified using the Failsafe PCR system (Premix E, Epicentre) using the following forward primer: 5΄-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3΄, and one of the following reverse primers (Illumina, indexed sequence is shown in bold font): 5΄-CAAGCAGAAGACGGCATACGAGAT CGTGAT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT ACATCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT GCCTAA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT TGGTCA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT CACTGT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, or 5΄-CAAGCAGAAGACGGCATACGAGAT ATTGGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄.

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements
    Article Snippet: The oligonucleotide was annealed by heating to 95 °C for 5 min in the presence of Buffer 2 (NEB) and 0.2 mM dNTPs, then cooled on ice for 3 min, thus creating a partially double stranded structure and internally primed substrate for DNA polymerase. .. This hairpin was PAGE purified on a 12 % non-denaturing gel.

    Polymerase Chain Reaction:

    Article Title: Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders
    Article Snippet: .. To the remaining 45 μl PCR mix, 5 μl of Buffer 2 (NEB) was added and the reactions were heated at 95°C for 10 minutes in a PCR machine and cooled slowly to 25°C to produce heteroduplexes. .. Each reaction was split in half and to one half 1 μl of T7E1 (NEB) was added.

    Article Title: Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis
    Article Snippet: .. Each DNA sample was sheared by passing it through a 30-gauge needle 65 times, and a 4-μg portion of each sheared DNA sample was added to a PCR tube containing 3 μg of M. catarrhalis genome-directed primers ( ) in 20 μl of buffer 2 (New England BioLabs, Ipswich, MA). .. This mixture was heated to 97°C for 3 min and 50 s and then immediately plunged into an ice water bath for 3 min. A 3-μl portion of Cy3-dCTP (1 mM) (GE Healthcare, Pittsburgh, PA) was added to 5 μl of a deoxynucleoside triphosphate (dNTP) mix containing 2 mM (each) dATP, dTTP, and dGTP combined with 1 mM dCTP.

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours. .. M.SssI was heat-inactivated at 65°C for 20 minutes, and removed by a PCR purification kit (Qiagen).

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Size selection following the ligation and PCR steps was performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads respectively (Beckman Coulter - A63880).

    Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum
    Article Snippet: The restriction reaction contained 2 units of Eco RI, 2 units of Mse I, 1.25 μL of buffer 2 (NEB), 100 ng·μL−1 BSA, and 250 ng of DNA in a final volume of 12.5 μL and was incubated for 3 h at 37°C. .. Each PCR contained 40 ng of each primer, 400 μmol of dNTPs, 1× Taq DNA polymerase buffer, 1 unit of Taq DNA polymerase (Invitrogen), and 1 μL of the ligation products in a final volume of 25 μL.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Article Title: Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing
    Article Snippet: RNA was hydrolyzed with 0.1 M KOH and heating at 95°C for 15 min, then reactions were cooled to room temperature and neutralized by adding HCl to 0.1 M. The cDNA was purified with DNA Clean & Concentrator columns and extended with Klenow DNA polymerase I (NEB) using the following primer: 5΄-ACACGACGCTCTTCCGATCTNNNNNNNN-Phos-3΄, in Buffer 2 (NEB) with 4 mM DTT at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. .. The tagged cDNA was purified with DNA Clean & Concentrator columns (Zymo) and amplified using the Failsafe PCR system (Premix E, Epicentre) using the following forward primer: 5΄-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3΄, and one of the following reverse primers (Illumina, indexed sequence is shown in bold font): 5΄-CAAGCAGAAGACGGCATACGAGAT CGTGAT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT ACATCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT GCCTAA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT TGGTCA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, 5΄-CAAGCAGAAGACGGCATACGAGAT CACTGT GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄, or 5΄-CAAGCAGAAGACGGCATACGAGAT ATTGGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3΄.

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: .. PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×. .. Diluted PCR amplicons were then heat denatured at 95 °C and cooled down to 20 °C with a 0.1 °C/s ramp.

    Article Title: A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish
    Article Snippet: T7 endonuclease I assay For the T7 endonuclease I assay, 10 μl of non-purified PCR product (ca. .. The re-annealed DNAs were then treated with 5 U (0.5 μl) of T7 endonuclease I (New England Biolabs) upon addition of 1.5 μl buffer 2 (New England Biolabs) and 3.5 μl H2 O to a final volume of 15 μl.

    Polyacrylamide Gel Electrophoresis:

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements
    Article Snippet: The oligonucleotide was annealed by heating to 95 °C for 5 min in the presence of Buffer 2 (NEB) and 0.2 mM dNTPs, then cooled on ice for 3 min, thus creating a partially double stranded structure and internally primed substrate for DNA polymerase. .. This hairpin was PAGE purified on a 12 % non-denaturing gel.

    IA:

    Article Title: RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation
    Article Snippet: After heatinactivation (80 o C for 15 min), NbBC IA (5U) was added during 1 h. On ice, streptavidin-coated magnetic beads (100 ng) were used to pull down the biotinylated primer PCR2a. .. After the supernatant was replaced by 100 μl buffer 2 (NEB) containing primer 3 (100 nM), and dNTPs (10 mM each) the sample was heated (70 o C for 20 min) to inactivate the enzyme and release the nicked hairpins from the beads.

    T7EI Assay:

    Article Title: Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders
    Article Snippet: Paragraph title: T7 Endonuclease I assay ... To the remaining 45 μl PCR mix, 5 μl of Buffer 2 (NEB) was added and the reactions were heated at 95°C for 10 minutes in a PCR machine and cooled slowly to 25°C to produce heteroduplexes.

    Article Title: A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish
    Article Snippet: Paragraph title: T7 endonuclease I assay ... The re-annealed DNAs were then treated with 5 U (0.5 μl) of T7 endonuclease I (New England Biolabs) upon addition of 1.5 μl buffer 2 (New England Biolabs) and 3.5 μl H2 O to a final volume of 15 μl.

    Chromatin Immunoprecipitation:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Replicate 2 of the H3K122ac and H3K64ac ChIPs, 2 replicates of H3K27ac ChIPs and all ChIP and input samples prepared from K562 cells were sequenced at Edinburgh Genomics (The University of Edinburgh, 50 base single end reads).

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×. .. Samples were finally run on a 2.5% agarose gel or on a BioAnalyzer chip (Agilent) to assess editing efficiency.

    Plasmid Preparation:

    Article Title: RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation
    Article Snippet: Restriction sites on the Adapter A were used to process the selected sequences into 19–20 bp inverted repeats, which were ligated into a pRETRO SUPER vector. .. After the supernatant was replaced by 100 μl buffer 2 (NEB) containing primer 3 (100 nM), and dNTPs (10 mM each) the sample was heated (70 o C for 20 min) to inactivate the enzyme and release the nicked hairpins from the beads.

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: .. Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells. .. Transformants were screened by colony PCR using insert‐specific primers, and insert integrity was confirmed by sequencing.

    Selection:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Size selection following the ligation and PCR steps was performed with 1x and 0.8x reaction volumes of Agencourt AMPure XP beads respectively (Beckman Coulter - A63880).

    Agarose Gel Electrophoresis:

    Article Title: RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation
    Article Snippet: After the supernatant was replaced by 100 μl buffer 2 (NEB) containing primer 3 (100 nM), and dNTPs (10 mM each) the sample was heated (70 o C for 20 min) to inactivate the enzyme and release the nicked hairpins from the beads. .. The fragments were separated (4.5% agarose gel) and 91 bp fragments were isolated from the gel by freeze squeezing and phenol extraction.

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×. .. Samples were finally run on a 2.5% agarose gel or on a BioAnalyzer chip (Agilent) to assess editing efficiency.

    In Vitro:

    Article Title: DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate
    Article Snippet: Paragraph title: In Vitro Methylation ... Briefly, plasmids were incubated with the M.Sssi (1 unit/μg DNA) in the presence of 1× Buffer 2 (New England Biolabs, Ipswich, MA) and 160 μmol/L SAM at 37°C for 4 hours.

    Article Title: Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA
    Article Snippet: E. coli tRNAMet was synthesized in vitro as previously described ( ) using the following protocol. .. The E. coli tRNAMet DNA template was synthesized in a 2-ml reaction containing 4 μM EctRNAMet 5 and EctRNAMet 3 primers, 400 μM deoxynucleoside triphosphates, 50 U of Klenow exo− DNA polymerase, and 1× buffer 2 from New England BioLabs.

    Transgenic Assay:

    Article Title: Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes
    Article Snippet: Paragraph title: Generation of transgenic tomato lines for complementation testing ... Purified PCR product was treated with T4 DNA polymerase (NEB, Ipswich, MA, USA) in 1 × Buffer 2 (NEB) and 2.5 m m dCTP at 22 °C for 30 min. To prepare the vector, pL2 was digested with Sna BI, purified and treated with T4 DNA polymerase in 1 × Buffer 2 and 2.5 m m dGTP at 22 °C for 30 min. PCR products (75 ng) and vector (50 ng) were incubated for 20 min at 75 °C, cooled at 4 °C, mixed together, the volume adjusted to 10 μL with water, and incubated at 65 °C for 1 min, and then 22 °C for 20 min, to anneal the complementary vector and insert overhangs prior to heat shock transformation of Escherichia coli Mach1 cells.

    Quantitation Assay:

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer
    Article Snippet: .. Quantitation of nucleosides by HPLC Genomic DNA was extracted with Gene Jet Genomic DNA extraction Kit (Thermo Fisher) or TRI Reagent (Sigma Aldrich), incubated with RNase A/T1 (Thermo Fisher) in buffer 2 (NEB), phenol/chloroform extracted and precipitated with ethanol. ..

    Concentration Assay:

    Article Title: Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins
    Article Snippet: .. PCR products were diluted by a factor 2 and complemented with Buffer 2 (New England Biolabs) to a final concentration of 1×. .. Diluted PCR amplicons were then heat denatured at 95 °C and cooled down to 20 °C with a 0.1 °C/s ramp.

    High Throughput Screening Assay:

    Article Title: Histone H3 globular domain acetylation identifies a new class of enhancers
    Article Snippet: After A-tailing reaction, enzymes were inactivated by incubation at 75 ºC for 20 min. and the ligation reaction was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1x buffer 2 (NEB), 7.5 % PEG-6,000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 16 °C overnight. .. Replicate 1 of the H3K122ac and H3K64ac ChIPs was sequenced at The Danish National High-Throughput DNA sequencing Center (Copenhagen; 42 base single end reads).

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