nebuffer 2  (New England Biolabs)


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    Name:
    NEBuffer 2
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    Catalog Number:
    B7002S
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs nebuffer 2

    https://www.bioz.com/result/nebuffer 2/product/New England Biolabs
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    nebuffer 2 - by Bioz Stars, 2019-12
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Complementary oligo sgRNAs ( ) were cloned into Bsm BI restriction site of LentiCRISPRv2 [ ] (Addgene ID 49535) that was used for lentivirus particle production. .. Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%).

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: The PCR products were cloned into pCR4 TOPO vector using a TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA), digested with KpnI and XhoI enzymes, and inserted into pGL4.15 luc2P/Hygro vector using T4 ligase as previously described [ ]. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Centrifugation:

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cells were washed in ice-cold PBS and 1–2 × 106 fixed cell pellets were used for the following steps. .. Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Triton X-100 was subsequently added to a 2% final concentration, and samples were incubated for 1 h at 37 °C.

    Amplification:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. .. Ligation was performed in 1x T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 1 mM ATP, 10 mM DTT, pH 7.5) with 1 μL of 400 U/μL T4 DNA ligase (NEB) in 60 μL total volume for 16 h at room temperature, then Q-column purified.

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations
    Article Snippet: Next, one-fifth of each PCR mixture was incubated in 15-μl reactions with 1× NEBuffer 2 and 5 U of T7EI (both from New England Biolabs). .. After 15 min. at 37°C, the samples were subjected to electrophoresis through 2% (w/v) agarose gels in 1× Tris–acetate–EDTA (TAE) buffer.

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies), and after adapter ligation (Illumina PE adapter with T4 DNA ligase; NEB) the bead-bound Hi-C DNA was amplified with seven PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Two step PCR amplification was as follows: primary denaturation at 99°C for 30 sec, denaturation at 99°C for 10 sec, annealing and extension at 70°C or 72°C for 8 min for W748S and E1143G mutations respectively and the final extension at 72°C for 10 min. Denaturation and annealing/extension steps were repeated for 30 times. .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Human DLEC1 promoter was amplified from genomic DNA isolated from HT29 cells using the following primers: 5′- GAC ACA AAT GTT TAC AAT GAC C-3′ (forward) and 5′- TTT CTC AAC TGC AGC CCC AGA T-3′ (reverse). .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes. .. Sequencing adapters (custom synthesized by IDT) were ligated to DNA using T/A ligation by incubating 10 µL DNA with 2 µL T4 DNA ligation buffer, 1 µL T4 ligase (NEB product number M020S), 40 µL of 40 µM pre-annealed adapter mix and 6 µL nuclease free water for 15 minutes at 20°C followed by 65°C at 10 minutes to deactivate the DNA ligase.

    Reporter Assay:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Paragraph title: Plasmids, transfection, and luciferase reporter assay ... Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    DNA Ligation:

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: We performed end repair by incubating each sample of DNA with 5 µL T4 DNA ligation buffer (New England Biolabs [NEB] product number B0202S), 4 µL of 10 mM dNTPs, 2.5 µL T4 DNA polymerase (NEB product number M0203S), 0.5 µL Klenow large fragment (NEB product number M0210S), 2.5 µL T4 PNK (NEB product number M0201S), and 5.5 µL nuclease free water for 30 minutes at 20°C. .. Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    Synthesized:

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: The RNA was ethanol precipitated, and first strand cDNA was synthesized using random hexamers and Superscript III (Invitrogen), according to manufacturer's protocol. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes. .. Following incubation, DNA was purified using a QIAGEN MinElute kit (product number 28004) to a final volume of 11 µL.

    TA Cloning:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: The PCR products were cloned into pCR4 TOPO vector using a TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA), digested with KpnI and XhoI enzymes, and inserted into pGL4.15 luc2P/Hygro vector using T4 ligase as previously described [ ]. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Blocking Assay:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr. .. We then added 1 μl of T7 endonuclease to each reaction, incubated at 37° for 15 min, added 2 μl of 0.25 mM EDTA to stop the reaction, and immediately loaded the entire reaction volume into a 1.5% agarose gel.

    Incubation:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: For all selections, the TALEN-digested library was incubated with 1 μL of 100 μg/μL RNase A (Qiagen) for 2 min and then Q-column purified. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. .. 50 μL of the eluted DNA was ligated with 2 pmol of heated and cooled #1 adapters containing barcodes corresponding to each sample (selections with different TALEN concentrations or constructs) ( ).

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: The cDNA was end repaired in a 100 μl reaction with 0.4 mM dNTPs, 10 μl 10× T4 DNA ligase buffer (NEB), 150 U ml-1 T4 DNA Polymerase (NEB), 50 U ml-1 T4 PNK (NEB), and 50 U ml-1 Klenow (NEB) for 30 min at 20 °C. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C. .. To prepare adapters, 40 μM universal adapter and 40 μM indexed adapter were mixed with 2 μl 10× primer annealing buffer (100 mM Tris-HCl pH 8, 50 mM NaCl) in a 20 μl reaction and heated at 95 °C for 15 min, 70 °C for 15 min, and slow cooled to room temperature.

    Article Title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations
    Article Snippet: Subsequently, the resulting amplicons were denaturated and slowly re-annealed by applying the program indicated in Supplementary Table S5. .. Next, one-fifth of each PCR mixture was incubated in 15-μl reactions with 1× NEBuffer 2 and 5 U of T7EI (both from New England Biolabs). .. After 15 min. at 37°C, the samples were subjected to electrophoresis through 2% (w/v) agarose gels in 1× Tris–acetate–EDTA (TAE) buffer.

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr. .. We then added 1 μl of T7 endonuclease to each reaction, incubated at 37° for 15 min, added 2 μl of 0.25 mM EDTA to stop the reaction, and immediately loaded the entire reaction volume into a 1.5% agarose gel.

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cells were washed in ice-cold PBS and 1–2 × 106 fixed cell pellets were used for the following steps. .. Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Triton X-100 was subsequently added to a 2% final concentration, and samples were incubated for 1 h at 37 °C.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: After the hybridisation incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer’s instructions (SureSelect Target Enrichment; Agilent Technologies). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: The digested DNA pieces were gel purified with QIAquick Gel Extraction Kit (Qiagen). .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs). .. 30 μg of pBABEpuro vector was digested with 0,5 U/ 1μg of plasmid DNA of SnaBI restriction enzyme (New England Biolabs) with 1 μg/ml of bovine serum albumin (BSA) (New England Biolabs) in NEBuffer 4. pBABEpuro was dephosphorylated with 0,5 U/1 μg of plasmid DNA of calf intestinal alkaline phosphatase (CIP) (Finnzymes) for one hour at 37°C and subsequently gel purified.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Following incubation, DNA was purified using a QIAGEN PCR-clean up kit. .. Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    TALENs:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: For all CCR5A and ATM TALENs, 6 μL of in vitro transcription/translation left TALEN and 6 μL of right TALEN were used, corresponding to a final concentration in a cleavage reaction of 16 nM ± 2 nM or 12 nM ± 1.5 nM for CC5A or ATM TALENs, respectively. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Modification:

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit

    Western Blot:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: These TALEN concentrations were quantified by Western blot performed in parallel with digestion. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Lysis:

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: The different tissues collected from piglets were digested in lysis buffer (0.4 M NaCl, 2 µM EDTA, 1% SDS, 10 µM Tris-HCl, and 100 µg/mL Proteinase K) overnight. .. A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler.

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cells were washed in ice-cold PBS and 1–2 × 106 fixed cell pellets were used for the following steps. .. Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Triton X-100 was subsequently added to a 2% final concentration, and samples were incubated for 1 h at 37 °C.

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Gel Purification:

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Countercurrent Chromatography:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Human DLEC1 promoter was amplified from genomic DNA isolated from HT29 cells using the following primers: 5′- GAC ACA AAT GTT TAC AAT GAC C-3′ (forward) and 5′- TTT CTC AAC TGC AGC CCC AGA T-3′ (reverse). .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Transfection:

    Article Title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations
    Article Snippet: To this end, genomic DNA samples from transfected or transduced cells were subjected to PCR for amplifying DNA segments encompassing the target sites of the different designer nucleases. .. Next, one-fifth of each PCR mixture was incubated in 15-μl reactions with 1× NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. PCR products were analyzed by Sanger sequencing and insertion or deletion (Indel) rates were assessed by the TIDE approach [ ] using DNA of non-target control cells as a reference control.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Paragraph title: Plasmids, transfection, and luciferase reporter assay ... Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Chromatography:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Ligation:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Briefly, to prepare purified nuclei for in situ ligation, we fixed five million cells as single-cell suspensions in growth medium with 2% formaldehyde for 30 min at room temperature, and then washed them one time in ice-cold 1× PBS. .. We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration.

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. .. 50 μL of the eluted DNA was ligated with 2 pmol of heated and cooled #1 adapters containing barcodes corresponding to each sample (selections with different TALEN concentrations or constructs) ( ).

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Samples were further incubated overnight at 37 °C with shaking after the addition of Hin dIII.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies), and after adapter ligation (Illumina PE adapter with T4 DNA ligase; NEB) the bead-bound Hi-C DNA was amplified with seven PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. The fragments were designed to include a short stretch of nonrandom DNA sequence at either end, which could be used as PCR primer binding sites, but no such PCR was performed as part of the experiments described here, and these nonrandom ends were removed as a consequence of the BamHI digestion step.

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: DNA was prepared for Illumina sequencing by shearing, end-repair and ligation. .. Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    Protease Inhibitor:

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cells were washed in ice-cold PBS and 1–2 × 106 fixed cell pellets were used for the following steps. .. Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Triton X-100 was subsequently added to a 2% final concentration, and samples were incubated for 1 h at 37 °C.

    Transferring:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Nick Translation:

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: Paragraph title: Genomic DNA Fragmentation and Nick Translation ... The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution.

    Infection:

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. The non-target control cells were transfected with the empty plasmid, that could not lead to genome editing.

    Generated:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr. .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Article Title: Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency
    Article Snippet: Genomic regions flanking the on-target as well as previously predicted off-target sites off-target sites for T7E1 assay were amplified using 100 ng of purified gDNA template, Q5 high-fidelity DNA polymerase (New England Biolabs) and specific primers (Integrated DNA Technologies, ) on a T100 thermal cycler (Bio-Rad). .. The PCR products generated using Q5 high-fidelity DNA polymerase were hybridized in NEBuffer2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) (New England Biolabs) by heating to 98 °C for 10 minutes, followed by a 2 °C/s ramp down to 85 °C, 1 min at 85 °C, and a 0.1 °C/s ramp down to 25 °C on a T100 thermal cycler (Bio-Rad). .. Subsequently, the annealed samples were subjected to T7 Endonuclease I (New England Biolabs) digestion for 30 min, separated by a 2% agarose gel and quantified on ChemiDoc XRS (Bio-Rad) using Quantity One software.

    other:

    Article Title: Ternary DNA computing using 3 × 3 multiplication matrices †Electronic supplementary information (ESI) available: Gel electrophoresis analysis, the stepwise treatment of H1, the individual 3 × 3 multiplication matrices operation of H2 and H3 and examples of fluorescence spectra corresponding to the parallel computation of three multiplication tables. See D
    Article Snippet: NEBuffer2 was purchased from New England Biolabs Inc. (Ipswich, MA).

    DNA Sequencing:

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes. .. Sequencing adapters (custom synthesized by IDT) were ligated to DNA using T/A ligation by incubating 10 µL DNA with 2 µL T4 DNA ligation buffer, 1 µL T4 ligase (NEB product number M020S), 40 µL of 40 µM pre-annealed adapter mix and 6 µL nuclease free water for 15 minutes at 20°C followed by 65°C at 10 minutes to deactivate the DNA ligase.

    Polymerase Chain Reaction:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: For the following, the cDNA was purified between each step using a PCR purification column (Qiagen). .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations
    Article Snippet: Subsequently, the resulting amplicons were denaturated and slowly re-annealed by applying the program indicated in Supplementary Table S5. .. Next, one-fifth of each PCR mixture was incubated in 15-μl reactions with 1× NEBuffer 2 and 5 U of T7EI (both from New England Biolabs). .. After 15 min. at 37°C, the samples were subjected to electrophoresis through 2% (w/v) agarose gels in 1× Tris–acetate–EDTA (TAE) buffer.

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: The primers for amplifying the DMD targeted fragments are listed in . .. A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler. .. The PCR products were digested with T7EN1 (M0302L; New England Biolabs, Ipswich, MA, USA) for 30 min at 37 °C and separated by 2.5% agarose gel electrophoresis.

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr. .. We then added 1 μl of T7 endonuclease to each reaction, incubated at 37° for 15 min, added 2 μl of 0.25 mM EDTA to stop the reaction, and immediately loaded the entire reaction volume into a 1.5% agarose gel.

    Article Title: Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency
    Article Snippet: Genomic regions flanking the on-target as well as previously predicted off-target sites off-target sites for T7E1 assay were amplified using 100 ng of purified gDNA template, Q5 high-fidelity DNA polymerase (New England Biolabs) and specific primers (Integrated DNA Technologies, ) on a T100 thermal cycler (Bio-Rad). .. The PCR products generated using Q5 high-fidelity DNA polymerase were hybridized in NEBuffer2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) (New England Biolabs) by heating to 98 °C for 10 minutes, followed by a 2 °C/s ramp down to 85 °C, 1 min at 85 °C, and a 0.1 °C/s ramp down to 25 °C on a T100 thermal cycler (Bio-Rad). .. Subsequently, the annealed samples were subjected to T7 Endonuclease I (New England Biolabs) digestion for 30 min, separated by a 2% agarose gel and quantified on ChemiDoc XRS (Bio-Rad) using Quantity One software.

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: PCR products were denatured, then temperature was reduced to 25°C. .. Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. ImageJ 2.0 software was used to quantify the band intensities.

    Article Title: Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations
    Article Snippet: The T7EI cleavage assay was performed as described previously . .. Briefly, the PCR products were purified, denatured and then re-annealed in NEBuffer 2 (NEB) using a thermocycler. .. Hybridized PCR products were digested with T7 endonuclease 1 (NEB, M0302L) for 30 min at 37 °C and subjected to 2% agarose gel electrophoresis.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Mutated PCR products were purified from parental template with DpnI restriction endonuclease (New England Biolabs). .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. Klenow fragment (3′→5′ exo− ; NEB) was added once the mixture cooled to 37°C, and the resulting reaction mixture was allowed to incubate for 1 h. This resulted in the extension of the partially overlapping oligonucleotides, each using the other as the template, resulting in a library of double-stranded DNA (dsDNA) fragments with an AT-rich random sequence flanking tetO (48 random base pairs to one side and 30 to the other) and a BamHI restriction site immediately following the random sequence to either side.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: The PCR products were cloned into pCR4 TOPO vector using a TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA), digested with KpnI and XhoI enzymes, and inserted into pGL4.15 luc2P/Hygro vector using T4 ligase as previously described [ ]. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: Fragmentation of the genomic DNA to 2 – 4 kb allows for optimal template size for performing PCR in droplets. .. The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution.

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Following incubation, DNA was purified using a QIAGEN PCR-clean up kit. .. Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    Sonication:

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: After removal of biotin from unligated DNA ends, the DNA was sonicated (Covaris E220) to an average size of around 400 base pairs, and end-repaired using DNA Polymerase I (Large Klenow Fragment), T4 DNA polymerase, and T4 DNA polynucleotide kinase (all NEB). dATP was added to the 3′ ends of the DNA (using Klenow Fragment (3′ → 5′ exo−); NEB), and the DNA was subjected to double-sided SPRI bead size selection (AMPure XP beads; Beckman Coulter). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Binding Assay:

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. Klenow fragment (3′→5′ exo− ; NEB) was added once the mixture cooled to 37°C, and the resulting reaction mixture was allowed to incubate for 1 h. This resulted in the extension of the partially overlapping oligonucleotides, each using the other as the template, resulting in a library of double-stranded DNA (dsDNA) fragments with an AT-rich random sequence flanking tetO (48 random base pairs to one side and 30 to the other) and a BamHI restriction site immediately following the random sequence to either side.

    Hi-C:

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Paragraph title: Hi-C experiments ... Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: Paragraph title: Promoter capture Hi-C ... After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Cleavage Assay:

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: Paragraph title: 4.5. T7EN1 Cleavage Assay and Sequencing ... A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler.

    Article Title: Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency
    Article Snippet: Paragraph title: T7E1 Cleavage assay ... The PCR products generated using Q5 high-fidelity DNA polymerase were hybridized in NEBuffer2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) (New England Biolabs) by heating to 98 °C for 10 minutes, followed by a 2 °C/s ramp down to 85 °C, 1 min at 85 °C, and a 0.1 °C/s ramp down to 25 °C on a T100 thermal cycler (Bio-Rad).

    Article Title: Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations
    Article Snippet: Paragraph title: T7EI cleavage assay ... Briefly, the PCR products were purified, denatured and then re-annealed in NEBuffer 2 (NEB) using a thermocycler.

    DNA Extraction:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    RNA Sequencing Assay:

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: Paragraph title: RNA-Seq ... To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Methylation:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

    Mutagenesis:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: After each F1 cross produced eggs, we genotyped the F1 animal for the presence of a CRISPR/Cas9-induced mutation using T7 endonuclease [New England Biolabs (NEB), Ipswich, MA; M0302L]. .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Article Title: Engineering CRISPR-Cpf1 crRNAs and mRNAs to maximize genome editing efficiency
    Article Snippet: The PCR products generated using Q5 high-fidelity DNA polymerase were hybridized in NEBuffer2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) (New England Biolabs) by heating to 98 °C for 10 minutes, followed by a 2 °C/s ramp down to 85 °C, 1 min at 85 °C, and a 0.1 °C/s ramp down to 25 °C on a T100 thermal cycler (Bio-Rad). .. Subsequently, the annealed samples were subjected to T7 Endonuclease I (New England Biolabs) digestion for 30 min, separated by a 2% agarose gel and quantified on ChemiDoc XRS (Bio-Rad) using Quantity One software.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Paragraph title: Generation of POLG-retroviral vectors with and without mutation(s) ... To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Isolation:

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: After the hybridisation incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer’s instructions (SureSelect Target Enrichment; Agilent Technologies). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2. .. After a post-capture PCR (four amplification cycles using Illumina PE PCR 1.0 and PE PCR 2.0 primers), the Promoter CHi-C libraries were purified with AMPure XP beads (Beckman Coulter) and paired-end sequenced (HiSeq 1000, Illumina) at the Babraham Institute Sequencing Facility.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Human DLEC1 promoter was amplified from genomic DNA isolated from HT29 cells using the following primers: 5′- GAC ACA AAT GTT TAC AAT GAC C-3′ (forward) and 5′- TTT CTC AAC TGC AGC CCC AGA T-3′ (reverse). .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Magnetic Beads:

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Luciferase:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

    Size-exclusion Chromatography:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Briefly, following single-fly DNA isolation (Puregene Cell and Tissue Kit, 158388; Qiagen) we PCR amplified a region surrounding the gRNA target site using oligos 5′-ACGCTTTTGCTCAGCATTTT-3′ and 5′-GGCTGGGGATACCATTTCTT-3′ (95° for 2 min, 35 cycles of 95° for 20 sec, 57° for 25 sec, and 72° for 30 sec, with a final 2-min extension at 72°). .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Two step PCR amplification was as follows: primary denaturation at 99°C for 30 sec, denaturation at 99°C for 10 sec, annealing and extension at 70°C or 72°C for 8 min for W748S and E1143G mutations respectively and the final extension at 72°C for 10 min. Denaturation and annealing/extension steps were repeated for 30 times. .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Labeling:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Paragraph title: Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS) ... We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration.

    Purification:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Briefly, to prepare purified nuclei for in situ ligation, we fixed five million cells as single-cell suspensions in growth medium with 2% formaldehyde for 30 min at room temperature, and then washed them one time in ice-cold 1× PBS. .. We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration.

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: For all selections, the TALEN-digested library was incubated with 1 μL of 100 μg/μL RNase A (Qiagen) for 2 min and then Q-column purified. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified. .. 50 μL of the eluted DNA was ligated with 2 pmol of heated and cooled #1 adapters containing barcodes corresponding to each sample (selections with different TALEN concentrations or constructs) ( ).

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: For the following, the cDNA was purified between each step using a PCR purification column (Qiagen). .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: The primers for amplifying the DMD targeted fragments are listed in . .. A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler. .. The PCR products were digested with T7EN1 (M0302L; New England Biolabs, Ipswich, MA, USA) for 30 min at 37 °C and separated by 2.5% agarose gel electrophoresis.

    Article Title: Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations
    Article Snippet: The T7EI cleavage assay was performed as described previously . .. Briefly, the PCR products were purified, denatured and then re-annealed in NEBuffer 2 (NEB) using a thermocycler. .. Hybridized PCR products were digested with T7 endonuclease 1 (NEB, M0302L) for 30 min at 37 °C and subjected to 2% agarose gel electrophoresis.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: The digested DNA pieces were gel purified with QIAquick Gel Extraction Kit (Qiagen). .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs). .. 30 μg of pBABEpuro vector was digested with 0,5 U/ 1μg of plasmid DNA of SnaBI restriction enzyme (New England Biolabs) with 1 μg/ml of bovine serum albumin (BSA) (New England Biolabs) in NEBuffer 4. pBABEpuro was dephosphorylated with 0,5 U/1 μg of plasmid DNA of calf intestinal alkaline phosphatase (CIP) (Finnzymes) for one hour at 37°C and subsequently gel purified.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution. .. The Genomic DNA fragmentation reaction mix is incubated at 15°C for 90 minutes and the reaction stopped by adding 37.5 μL of 0.5 M EDTA, pH 8.0 (Ambion, AM9260G).

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Following incubation, DNA was purified using a QIAGEN PCR-clean up kit. .. Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    Sequencing:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Paragraph title: Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS) ... We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration.

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: For selections on CCR5A and ATM sequence libraries, the combined pre-selection library was further purified in a 300,000 MWCO spin column (Sartorius) with three 500-μL washes in 1x NEBuffer 3. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C. .. To A-tail the cDNA, 2 mM dATP, 32 μl cDNA, 5 μl 10× NEBuffer 2 (NEB), and 300 U ml-1 Klenow exo (3′ to 5′ exo minus) (NEB) was added to the cDNA in a 50 μl reaction, and incubated for 30 min at 37 °C.

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: Paragraph title: 4.5. T7EN1 Cleavage Assay and Sequencing ... A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler.

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. ImageJ 2.0 software was used to quantify the band intensities.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs). .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP).

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: Incorporation of biotinylated nucleotides during the nick translation step allows the genomic DNA to be removed during the Genomic DNA Removal step to reduce the amount of genomic DNA fragments entering into the construction of the Illumina GA II and Roche 454 sequencing libraries. .. The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution.

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Paragraph title: Sample preparation, sequencing, and bioinformatics of pooled samples ... Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    Selection:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: Paragraph title: In Vitro Selection for DNA Cleavage ... 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: After removal of biotin from unligated DNA ends, the DNA was sonicated (Covaris E220) to an average size of around 400 base pairs, and end-repaired using DNA Polymerase I (Large Klenow Fragment), T4 DNA polymerase, and T4 DNA polynucleotide kinase (all NEB). dATP was added to the 3′ ends of the DNA (using Klenow Fragment (3′ → 5′ exo−); NEB), and the DNA was subjected to double-sided SPRI bead size selection (AMPure XP beads; Beckman Coulter). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Construct:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

    CRISPR:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: Paragraph title: Ugt86Dd CRISPR/Cas9 editing ... Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Paragraph title: CRISPR-Cas9 engineering of targeted cells and functional screen ... Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%).

    Agarose Gel Electrophoresis:

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: PCR products were denatured, then temperature was reduced to 25°C. .. Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. ImageJ 2.0 software was used to quantify the band intensities.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. The fragments were designed to include a short stretch of nonrandom DNA sequence at either end, which could be used as PCR primer binding sites, but no such PCR was performed as part of the experiments described here, and these nonrandom ends were removed as a consequence of the BamHI digestion step.

    Titration:

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: Incorporation of biotinylated nucleotides during the nick translation step allows the genomic DNA to be removed during the Genomic DNA Removal step to reduce the amount of genomic DNA fragments entering into the construction of the Illumina GA II and Roche 454 sequencing libraries. .. The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution. .. The Genomic DNA fragmentation reaction mix is incubated at 15°C for 90 minutes and the reaction stopped by adding 37.5 μL of 0.5 M EDTA, pH 8.0 (Ambion, AM9260G).

    Chromatin Immunoprecipitation:

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Plasmid Preparation:

    Article Title: Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
    Article Snippet: A mixture of 50 ng wild-type purified PCR product and 150 ng of the tested purified PCR product was denatured and re-annealed in NEBuffer 2 (New England Biolabs, Ipswich, MA, USA) using a thermocycler. .. The PCR products were digested with T7EN1 (M0302L; New England Biolabs, Ipswich, MA, USA) for 30 min at 37 °C and separated by 2.5% agarose gel electrophoresis.

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%). .. PCR products were analyzed by Sanger sequencing and insertion or deletion (Indel) rates were assessed by the TIDE approach [ ] using DNA of non-target control cells as a reference control.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Inserts POLGα , POLGα W748S, POLGα E1143G and POLGα W748S E1143G were digested from 30 μg of plasmid in double digestion reactions with EcoRI and BamHI restriction endonucleases in EcoRI buffer (Fermentas). .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: The PCR products were cloned into pCR4 TOPO vector using a TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA), digested with KpnI and XhoI enzymes, and inserted into pGL4.15 luc2P/Hygro vector using T4 ligase as previously described [ ]. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Software:

    Article Title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations
    Article Snippet: Next, one-fifth of each PCR mixture was incubated in 15-μl reactions with 1× NEBuffer 2 and 5 U of T7EI (both from New England Biolabs). .. After 15 min. at 37°C, the samples were subjected to electrophoresis through 2% (w/v) agarose gels in 1× Tris–acetate–EDTA (TAE) buffer.

    Hybridization:

    Article Title: Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages
    Article Snippet: After the hybridisation incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer’s instructions (SureSelect Target Enrichment; Agilent Technologies). .. After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2.

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Functional Assay:

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein
    Article Snippet: Paragraph title: CRISPR-Cas9 engineering of targeted cells and functional screen ... Hybridized PCR products were digested with T7 endonuclease 1 (NEB) for 20 min at 37°C in NEBuffer 2 (NEB, USA) and analyzed by agarose gel electrophoresis (2%).

    Recombinant:

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).

    Sample Prep:

    Article Title: Genomic Evidence of Rapid and Stable Adaptive Oscillations over Seasonal Time Scales in Drosophila
    Article Snippet: Paragraph title: Sample preparation, sequencing, and bioinformatics of pooled samples ... Next, we performed dATP addition by incubating 32 µL of DNA with 5 µL 10× NEBuffer 2 (NEB product number B7002S), 1 µL 10 mM dATP, 3 µL Klenow Exo-minus (NEB product number M0212S), and 9 µL nuclease free water at 37°C for 30 minutes.

    In Vitro:

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: Paragraph title: In Vitro Selection for DNA Cleavage ... 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Ethanol Precipitation:

    Article Title: Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs
    Article Snippet: Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. .. Cell pellets were resuspended in 3 C lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.2% NP-40 pH 8.0) with 1 × protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2 × NE Buffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking.

    Produced:

    Article Title: Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster
    Article Snippet: After each F1 cross produced eggs, we genotyped the F1 animal for the presence of a CRISPR/Cas9-induced mutation using T7 endonuclease [New England Biolabs (NEB), Ipswich, MA; M0302L]. .. Subsequently 10 μl of PCR product was mixed with 7 μl of molecular grade water and 2 μl of NEB buffer 2 (B7002S), incubated at 95° for 5 min in a heat block, and allowed to slowly cool to room temperature for ∼2 hr.

    Concentration Assay:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: To prepare single-nucleus suspensions, we first lysed fixed cells in a buffer containing 10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40 pH 8 for 90 min at 4 °C, and then in a buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.3% SDS pH 8 for 45 min at 37 °C. .. We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration. .. We mildly rotated cells for a short time at 37 °C (8 min for HeLa, 4 min for mouse B-lymphocytes), after which we transferred them onto ice.

    Article Title: Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity
    Article Snippet: For all CCR5A and ATM TALENs, 6 μL of in vitro transcription/translation left TALEN and 6 μL of right TALEN were used, corresponding to a final concentration in a cleavage reaction of 16 nM ± 2 nM or 12 nM ± 1.5 nM for CC5A or ATM TALENs, respectively. .. 50 μL of purified DNA was incubated with 3 μL of 10 mM dNTP mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP) (NEB), 6 μL of 10x NEBuffer 2, and 1 μL of 5 U/μL Klenow Fragment DNA Polymerase (NEB) for 30 min at room temperature and Q-column purified.

    Article Title: Chromatin modification mapping in nanochannels
    Article Snippet: Chromatin was reconstituted from calf thymus (Sigma H9250), HeLa core (Activ Motif 53501), and chicken erythrocyte (Genway Biotech, Inc 11-511-248355) histones. .. 6 μ l of T4 GT7 DNA was dispersed in a buffer containing 20 μ l of 10× NEBuffer2 (New England Biolabs), 44.5 μ l of 0.1 M Na EDTA, 236.8 μ l of 4 M NaCl, 10 μ l of 20 mg/ml bovine serum albumin (BSA) in 152 μ l of ultrapure water to give a final NaCl concentration of 2 M. For the reconstitution of calf thymus, HeLa core, and chicken erythrocyte chromatin, the histones of calf thymus, HeLa core, and chicken erythrocytes, were added at 2 μ g, 6 μ g, and 10 μ g, respectively. .. The different histone concentrations were necessary to obtain mechanically similar chromatin as determined by nanochannel stretching and molecular combing assays that we performed in exploratory stages of this work.

    Article Title: Synthetic Promoters Functional in Francisella novicida and Escherichia coli
    Article Snippet: VgrG is a 17.5-kDa F. novicida virulence factor that is part of the type VI secretion system encoded by the Francisella pathogenicity island (FPI) ( ). .. Oligonucleotides BamHI-N48- tetO and BamHI-N30- tetO rc ( ) were added to a final concentration of 2 μM in 1× NEBuffer 2 (NEB) with 250 μM each deoxynucleoside triphosphate (dNTP). .. The mixture was brought to a boil and then allowed to cool slowly to facilitate the annealing together of the two oligonucleotides at their complementary tetO regions, which overlap each other by the full 19 nt of tetO .

    Article Title: Microdroplet-based PCR amplification for large scale targeted sequencing
    Article Snippet: Incorporation of biotinylated nucleotides during the nick translation step allows the genomic DNA to be removed during the Genomic DNA Removal step to reduce the amount of genomic DNA fragments entering into the construction of the Illumina GA II and Roche 454 sequencing libraries. .. The genomic DNA samples are processed on ice: 7.8 μg of genomic DNA in 25 μL nuclease free water (Fisher, 50843418), 37.5 μL of NEBuffer 2 (NEB, B7002S), 37.5 μL of a mixture of 1 mM each dCTP, dGTP, dTTP (NEB, N0446S), 18.75 μL of 0.4 mM Biotin-14 dATP (Invitrogen, 19524-016), 211.25 μL nuclease free water (Fisher, 50843418), 7.5 μL DNA polymerase I (10 units/μL) (NEB, M0209S), 37.5 μL of DNase I (NEB, M0303S) at a concentration determined by titration to achieve a 2-4 kb size distribution. .. The Genomic DNA fragmentation reaction mix is incubated at 15°C for 90 minutes and the reaction stopped by adding 37.5 μL of 0.5 M EDTA, pH 8.0 (Ambion, AM9260G).

    In Situ:

    Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing
    Article Snippet: Briefly, to prepare purified nuclei for in situ ligation, we fixed five million cells as single-cell suspensions in growth medium with 2% formaldehyde for 30 min at room temperature, and then washed them one time in ice-cold 1× PBS. .. We resuspended lysed cells in 1× NEBuffer 2 (NEB) supplemented with 0.1% Triton X-100 and Proteinase K at 100 μg/ml final concentration.

    Gel Extraction:

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: The digested DNA pieces were gel purified with QIAquick Gel Extraction Kit (Qiagen). .. To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs).

    Article Title: Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq
    Article Snippet: FA wash buffer 3 10 m M Tris–Cl, pH 8.0 25 m M LiCl 1.0% (v/v) IGEPAL 1.0% (w/v) Sodium deoxycholate 2 m M EDTA, pH 8.0 Filter 0.22 μm. .. ChIP buffers FA lysis buffer+0.2% (w/v) SDS 10 m M Tris–HCl, pH 7.5 Antibody Anti-histone H3 antibody—Abcam, catalog #ab1791 Enzymes T4 DNA polymerase (NEB) T4 DNA ligase (NEB) Phi29 DNA polymerase (NEB) Klenow fragment exo- (NEB) Taq DNA polymerase (NEB) Enzyme buffers 10 × NEBuffer 2 (NEB) 10 × T4 DNA ligase buffer (NEB) 10 × Phi29 polymerase buffer (NEB) 10 × Taq polymerase buffer (NEB) Aliquot stocks 20% (w/v) SDS 3 m M dNTPs 25 m M dNTPs 3 m M dATP 1 × BSA (1 mg/ml) CPI stock Adaptors and primers Sequencing adaptors (15 μ M ) PCR primers (20 μ M ) Index (20 μ M ) Other Magna ChIP Protein A Magnetic Beads (Millipore) DynaMag—15 magnet—Invitrogen, catalog #123-01D DynaMag—2 magnet—Invitrogen, catalog #123-21D Agarose, see above Qiagen QIAquick Gel Extraction Kit .. Thaw the MNase-digested supernatant from — 80 °C storage.

    Article Title: Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1
    Article Snippet: To further generate the methylated luciferase reporter, the constructs were treated with methyl-trasferase M. SssI. .. Briefly, 5 μg reporter constructs were incubated with 5 unites of M. SssI in NEBuffer 2 (New England Biolabs Inc. Ipswich, MA, USA) supplemented with 160 μM S-adenosylmethionine (SAM, New England Biolabs Inc) at 37°C for 1 h. After the reaction, the methylated luciferase reporter plasmids were purified by gel extraction using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). .. The methylation-dependent HhaI and HpaII restriction endonucleases were used to confirm the efficiency of the methylation reaction.

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