neb3 buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs neb3 buffer
    Neb3 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neb3 buffer/product/New England Biolabs
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    neb3 buffer - by Bioz Stars, 2019-12
    94/100 stars

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    Related Articles

    Centrifugation:

    Article Title: ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis
    Article Snippet: Mouse anti-?Actin (Sigma-Aldrich, St.Louis, MO, USA) or rabbit anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA) were used as loading controls. .. For dephosphorylation experi-ments, H2 O2 treated or untreated HeLa cells were harvested by trypsinization, collected by centrifugation at 170g for 10 min, washed twice with PBS and resuspended in NEB3 Buffer (New England Biolabs, Ipswich, MA, USA), in which Calf intestinal phosphatase (CIP; New England Biolabs, Ipswich, MA, USA) has optimal activity. .. Cleared lysates were split and one half was incubated with 10U CIP for 30 min at 37°C, whereas the other half was mock treated.

    Amplification:

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [ ]. .. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight.

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere. .. The nuclei were incubated in the same buffer but with 5U T4 DNA ligase (Thermo Scientific) at 16°C at 50 rpm for 4.5 h, and then for 30 min at room temperature.

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: T4 DNA ligase (New England Biolabs, cat. no.M0202) T4 DNA Ligase Buffer 10× (New England Biolabs, cat. no. B0202S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201) Deoxyribonucleotide triphosphates (dNTPs; 10 mM each nucleotide; New England Biolabs, cat. no. N0447) DpnI restriction endonuclease (New England Biolabs, cat. no. R0176) Taq polymerase (New England Biolabs, cat. no. M0273) Phusion® High-Fidelity DNA polymerase (New England Biolabs, cat. no. M0530S) ▲CRITICAL – a high-fidelity polymerase should be used for amplification products intended for use in downstream deep-sequencing to limit PCR errors. .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication ( , ), can explain an occurrence of large amounts of double-stranded DNA following a RCR process. .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: ・ QIAquick PCR Purification Kit (Qiagen) ・ QIAquick Gel Extraction kit (Qiagen) ・ Primer pairs for amplification of CNEs: 10 pmol/μL in TE (see Methods for details), stored at −20°C. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

    Whole Genome Amplification:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere. .. The nuclei were incubated in the same buffer but with 5U T4 DNA ligase (Thermo Scientific) at 16°C at 50 rpm for 4.5 h, and then for 30 min at room temperature.

    Recovery:

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Pyrolysis Gas Chromatography:

    Article Title: N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils
    Article Snippet: The PGC was subsequently washed with 2 mL of 50 mM triethylamine acetate (TEAA) buffer, pH 7.0. .. Dried material was reconstituted in 85 µL of MilliQ water before 10 µL of 10× NEB3 buffer and 5 µL (50 U) of calf intestinal phosphatase (both New England Biolabs) were added.

    Blocking Assay:

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: For the following, generic reagents and equipment are likely to suffice. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge .. The following sections describe how to identify and analyze enhancers in Xenopus , using as an example our analysis of the 5’ flanking region of a paired domain homeobox gene, pax6 ( ).

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: To demonstrate that the upper RPB1 band detected in immunoblots with anti-HA or anti-RPB1 antibodies corresponded to phosphorylated RPB1, 8 × 107 parasites were resuspended in 25 μl of lysis buffer (2% SDS, 100 mM Tris-HCl [pH 6.8]) and boiled for 5 min. To enable alkaline phosphatase treatment, the lysate was diluted 20-fold with Tryp wash solution (100 mM NaCl, 3 mM MgCl2 , 20 mM Tris-HCl [pH 7.5]) to reduce the SDS concentration to 0.05% and subsequently reduced to a volume of 100 μl by using an Amicon Ultra 0.5-ml filter device (Millipore). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture. .. The research group of C. C. Wang (University of California, San Francisco) had previously studied all 11 T. brucei CRKs by RNAi-mediated gene knockdown.

    SYBR Green Assay:

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Incubation:

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
    Article Snippet: For 3C analysis cells were crosslinked and digested as described for ChIP . .. Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking. .. Digestion was checked loading digested and undigested controls on a 0.6% agarose gel.

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [ ]. .. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight. .. The cut PCR products were analyzed by 8% acrylamide gel and SYBR stain (Lonza, USA).

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere. .. Pronuclei were washed once in 1× DpnII buffer (NEB) with 2× BSA (NEB), and then, chromatin was digested overnight in 9 μl of the same solution but with 5 U DpnII (NEB).

    Article Title: Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD
    Article Snippet: GammaBind Plus Sepharose beads were resuspended in RIPApi buffer, and 30 µl of this mixture was added to the ovary extracts and incubated for 1.5 h at 4°C with constant mixing. .. For phosphatase treatment, beads were washed twice with wash buffer 1, once with wash buffer 2 (wash buffer 1 lacking phosphatase inhibitors) and once in a 1∶1 mixture of wash buffer 2 and NEB3 buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9; New England Biolabs).

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication ( , ), can explain an occurrence of large amounts of double-stranded DNA following a RCR process. .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: To demonstrate that the upper RPB1 band detected in immunoblots with anti-HA or anti-RPB1 antibodies corresponded to phosphorylated RPB1, 8 × 107 parasites were resuspended in 25 μl of lysis buffer (2% SDS, 100 mM Tris-HCl [pH 6.8]) and boiled for 5 min. To enable alkaline phosphatase treatment, the lysate was diluted 20-fold with Tryp wash solution (100 mM NaCl, 3 mM MgCl2 , 20 mM Tris-HCl [pH 7.5]) to reduce the SDS concentration to 0.05% and subsequently reduced to a volume of 100 μl by using an Amicon Ultra 0.5-ml filter device (Millipore). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture. .. The research group of C. C. Wang (University of California, San Francisco) had previously studied all 11 T. brucei CRKs by RNAi-mediated gene knockdown.

    Activity Assay:

    Article Title: ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis
    Article Snippet: Mouse anti-?Actin (Sigma-Aldrich, St.Louis, MO, USA) or rabbit anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA) were used as loading controls. .. For dephosphorylation experi-ments, H2 O2 treated or untreated HeLa cells were harvested by trypsinization, collected by centrifugation at 170g for 10 min, washed twice with PBS and resuspended in NEB3 Buffer (New England Biolabs, Ipswich, MA, USA), in which Calf intestinal phosphatase (CIP; New England Biolabs, Ipswich, MA, USA) has optimal activity. .. Cleared lysates were split and one half was incubated with 10U CIP for 30 min at 37°C, whereas the other half was mock treated.

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: A plasmid carrying the gata2 basal promoter linked to the GFP-polyA , which is available from R. M. Grainger, may provide more sensitivity for the co-transgenesis assay since it appears to impart higher levels of transcriptional activity to enhancers without causing background expression on its own. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

    Expressing:

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: A plasmid carrying the gata2 basal promoter linked to the GFP-polyA , which is available from R. M. Grainger, may provide more sensitivity for the co-transgenesis assay since it appears to impart higher levels of transcriptional activity to enhancers without causing background expression on its own. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

    Western Blot:

    Article Title: ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis
    Article Snippet: For dephosphorylation experi-ments, H2 O2 treated or untreated HeLa cells were harvested by trypsinization, collected by centrifugation at 170g for 10 min, washed twice with PBS and resuspended in NEB3 Buffer (New England Biolabs, Ipswich, MA, USA), in which Calf intestinal phosphatase (CIP; New England Biolabs, Ipswich, MA, USA) has optimal activity. .. Cleared lysates were split and one half was incubated with 10U CIP for 30 min at 37°C, whereas the other half was mock treated.

    Article Title: Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD
    Article Snippet: Paragraph title: Small scale immunoprecipitations and western blotting ... For phosphatase treatment, beads were washed twice with wash buffer 1, once with wash buffer 2 (wash buffer 1 lacking phosphatase inhibitors) and once in a 1∶1 mixture of wash buffer 2 and NEB3 buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9; New England Biolabs).

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: To demonstrate that the upper RPB1 band detected in immunoblots with anti-HA or anti-RPB1 antibodies corresponded to phosphorylated RPB1, 8 × 107 parasites were resuspended in 25 μl of lysis buffer (2% SDS, 100 mM Tris-HCl [pH 6.8]) and boiled for 5 min. To enable alkaline phosphatase treatment, the lysate was diluted 20-fold with Tryp wash solution (100 mM NaCl, 3 mM MgCl2 , 20 mM Tris-HCl [pH 7.5]) to reduce the SDS concentration to 0.05% and subsequently reduced to a volume of 100 μl by using an Amicon Ultra 0.5-ml filter device (Millipore). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Hybridization:

    Article Title: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis
    Article Snippet: For labeling of genomic DNA, 1 μg of MboI-digested DNA was denatured for 5 min at 94°C followed by the addition of 9 μg of random primers (Invitrogen), 1× NEB3 buffer (New England Biolabs), 0.1 mg/ml BSA, 20 mM DTT, 10 units of the Klenow fragment of DNA polymerase (New England Biolabs), 40 μmol each of dATP, dTTP, and dGTP, 25 μmol of dCTP; 1.5 nmol of Cye dye-labeled dCTP (Amersham Pharmacia Biosciences), and water to a final volume of 100 μl. .. For labeling of genomic DNA, 1 μg of MboI-digested DNA was denatured for 5 min at 94°C followed by the addition of 9 μg of random primers (Invitrogen), 1× NEB3 buffer (New England Biolabs), 0.1 mg/ml BSA, 20 mM DTT, 10 units of the Klenow fragment of DNA polymerase (New England Biolabs), 40 μmol each of dATP, dTTP, and dGTP, 25 μmol of dCTP; 1.5 nmol of Cye dye-labeled dCTP (Amersham Pharmacia Biosciences), and water to a final volume of 100 μl.

    Inter Assay:

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight. .. The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight.

    Flow Cytometry:

    Article Title: N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils
    Article Snippet: The flow through of the C-18 column was collected and loaded onto the porous graphitic carbon (PGC) cartridge. .. Dried material was reconstituted in 85 µL of MilliQ water before 10 µL of 10× NEB3 buffer and 5 µL (50 U) of calf intestinal phosphatase (both New England Biolabs) were added.

    Ligation:

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
    Article Snippet: Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking. .. Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking.

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: A starting plasmid to generate libraries that does not contain sites for the type IIS endonuclease that you plan to use for the cassette ligation strategy, such as pRNDM ( ). .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Protease Inhibitor:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

    Hemagglutination Assay:

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: HA-tagged protein was detected with a commercial monoclonal rat anti-HA antibody (Roche), and RPA1 was detected with a polyclonal rabbit immune serum as described previously ( ). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Generated:

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: Conditional yeast strains can be generated de novo, or located in previously published work and requested. .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    other:

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
    Article Snippet: As controls, BACs corresponding to the region of interested were digested with 100 U BglII in NEB3 buffer in 50 μl o/n at 37 °C.

    Sequencing:

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: COBRA for LINE-1 was performed as previously described, the 5′UTR of LINE-1.2 (L1Hs) sequence from NCBI Accession Number M80343 was used . .. The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight.

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Sonication:

    Article Title: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis
    Article Snippet: For labeling of genomic DNA, 1 μg of MboI-digested DNA was denatured for 5 min at 94°C followed by the addition of 9 μg of random primers (Invitrogen), 1× NEB3 buffer (New England Biolabs), 0.1 mg/ml BSA, 20 mM DTT, 10 units of the Klenow fragment of DNA polymerase (New England Biolabs), 40 μmol each of dATP, dTTP, and dGTP, 25 μmol of dCTP; 1.5 nmol of Cye dye-labeled dCTP (Amersham Pharmacia Biosciences), and water to a final volume of 100 μl. .. After an incubation of 90 min at 37°C, the mixture was purified with the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France).

    Injection:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: To obtain G2‐phase data, zygotes were fixed 27 h post‐hCG injection (corresponding to about 15 h post‐fertilization) and lysed, and pronuclei were separated into different wells after SDS lysis according to their size. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

    Binding Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication ( , ), can explain an occurrence of large amounts of double-stranded DNA following a RCR process. .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Hi-C:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: Paragraph title: Single‐nucleus Hi‐C ... The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

    Nucleic Acid Electrophoresis:

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: For the following, generic reagents and equipment are likely to suffice. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge .. The following sections describe how to identify and analyze enhancers in Xenopus , using as an example our analysis of the 5’ flanking region of a paired domain homeobox gene, pax6 ( ).

    Combined Bisulfite Restriction Analysis Assay:

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [ ]. .. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight. .. The cut PCR products were analyzed by 8% acrylamide gel and SYBR stain (Lonza, USA).

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: COBRA for LINE-1 was performed as previously described, the 5′UTR of LINE-1.2 (L1Hs) sequence from NCBI Accession Number M80343 was used . .. The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight.

    Methylation:

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [ ]. .. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight.

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: In brief, the DNA samples were converted by a bisulphite reaction such that unmethylated cytosine (u C) would be converted to uracil (U), whereas methylated cytosine (m C) would remain as cytosine. .. The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight.

    Isolation:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

    Article Title: N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils
    Article Snippet: Paragraph title: Isolation of Nucleoside Diphospho Sugars. ... Dried material was reconstituted in 85 µL of MilliQ water before 10 µL of 10× NEB3 buffer and 5 µL (50 U) of calf intestinal phosphatase (both New England Biolabs) were added.

    Labeling:

    Article Title: Mass spectrometric analysis of purine de novo biosynthesis intermediates
    Article Snippet: Calf intestinal alkaline phosphatase (CIP) and NEB3 buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA), and Dulbecco's minimum essential medium (DMEM), F12 nutrient mix, and fetal bovine serum (FBS) were obtained from Life Technologies, ThermoFisher Scientific (MA, USA). .. Calf intestinal alkaline phosphatase (CIP) and NEB3 buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA), and Dulbecco's minimum essential medium (DMEM), F12 nutrient mix, and fetal bovine serum (FBS) were obtained from Life Technologies, ThermoFisher Scientific (MA, USA).

    Article Title: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis
    Article Snippet: For determination of the colony-forming units, 105 cells counted by flow cytometry and their dilution series were plated on selective medium. .. For labeling of genomic DNA, 1 μg of MboI-digested DNA was denatured for 5 min at 94°C followed by the addition of 9 μg of random primers (Invitrogen), 1× NEB3 buffer (New England Biolabs), 0.1 mg/ml BSA, 20 mM DTT, 10 units of the Klenow fragment of DNA polymerase (New England Biolabs), 40 μmol each of dATP, dTTP, and dGTP, 25 μmol of dCTP; 1.5 nmol of Cye dye-labeled dCTP (Amersham Pharmacia Biosciences), and water to a final volume of 100 μl. .. After an incubation of 90 min at 37°C, the mixture was purified with the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France).

    Purification:

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
    Article Snippet: Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking. .. Then the sample was incubated with 1.6% SDS for 25 min at 65 °C and with 1.15× ligation buffer (New England BioLabs) and 1% Triton X-100 for 1 h at 37 °C.

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: ・ QIAquick PCR Purification Kit (Qiagen) ・ QIAquick Gel Extraction kit (Qiagen) ・ Primer pairs for amplification of CNEs: 10 pmol/μL in TE (see Methods for details), stored at −20°C. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: For the generation of a polyclonal anti-RPB1 immune serum, the CTD portion of RPB1, comprising the C-terminal 228 amino acids, was expressed with an N-terminal glutathione S -transferase (GST) tag in Escherichia coli , purified by glutathione affinity chromatography, and used as a GST fusion protein to immunize rats according to a previously published protocol ( ). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Polymerase Chain Reaction:

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: To observe methylation levels of Alu in samples, the sodium-bisulfite-treated DNA in each sample was amplified by PCR containing 1x PCR buffer (Qiagen, Germany), 0.2 mM of deoxynucleotide triphosphate (Promega, USA), 1 mM of magnesium chloride (Qiagen, Germany), 25 U of HotStarTaq DNA Polymerase (Qiagen, Germany), and 0.3 μM primer pairs: ALU-BRev (5′-CTAACTTTTTATATTTTTAATAAAAACRAAATTTCAC CA-3′) where R = A and G and Y = C and T. For Alu amplification, the program was set as follows: 95 °C for 15 min, 40 cycles of 95 °C for 45 s, 57 °C for 45 s, and 72 °C for 45 s, followed by a final extension of 72 °C for 7 min [ ]. .. Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight. .. The cut PCR products were analyzed by 8% acrylamide gel and SYBR stain (Lonza, USA).

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: One microlitre of bisulphite DNA was then subjected to 35 cycles of PCR, at a 50°C annealing temperature using the following primer sets: LINE-1-F (5′-CCGTAAGGGGTTAGGGAGTTTTT-3′) and LINE-1-R (5′-RTAAAACCCTCCRAACCAAATATAAA-3′). .. The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight.

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: T4 DNA ligase (New England Biolabs, cat. no.M0202) T4 DNA Ligase Buffer 10× (New England Biolabs, cat. no. B0202S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201) Deoxyribonucleotide triphosphates (dNTPs; 10 mM each nucleotide; New England Biolabs, cat. no. N0447) DpnI restriction endonuclease (New England Biolabs, cat. no. R0176) Taq polymerase (New England Biolabs, cat. no. M0273) Phusion® High-Fidelity DNA polymerase (New England Biolabs, cat. no. M0530S) ▲CRITICAL – a high-fidelity polymerase should be used for amplification products intended for use in downstream deep-sequencing to limit PCR errors. .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. CAUTION Ethidium bromide is toxic and a DNA mutagen; handle properly and avoid contact using appropriate Personal Protective Equipment.

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: For the following, generic reagents and equipment are likely to suffice. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge .. The following sections describe how to identify and analyze enhancers in Xenopus , using as an example our analysis of the 5’ flanking region of a paired domain homeobox gene, pax6 ( ).

    De-Phosphorylation Assay:

    Article Title: ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis
    Article Snippet: Mouse anti-?Actin (Sigma-Aldrich, St.Louis, MO, USA) or rabbit anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA) were used as loading controls. .. For dephosphorylation experi-ments, H2 O2 treated or untreated HeLa cells were harvested by trypsinization, collected by centrifugation at 170g for 10 min, washed twice with PBS and resuspended in NEB3 Buffer (New England Biolabs, Ipswich, MA, USA), in which Calf intestinal phosphatase (CIP; New England Biolabs, Ipswich, MA, USA) has optimal activity. .. Cleared lysates were split and one half was incubated with 10U CIP for 30 min at 37°C, whereas the other half was mock treated.

    Lysis:

    Article Title: A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture
    Article Snippet: Briefly, after pronuclei were isolated, they were fixed in 2% formaldehyde for 15 min and then lysed on ice in lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.5% (v/v) NP‐40 substitute (Sigma), 1% (v/v) Triton X‐100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) for at least 15 min. .. The pronuclei were washed once through PBS and 1× NEB3 buffer (NEB) with 0.6% SDS, in which they were then incubated at 37°C for 2 h with shaking in humidified atmosphere.

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: To demonstrate that the upper RPB1 band detected in immunoblots with anti-HA or anti-RPB1 antibodies corresponded to phosphorylated RPB1, 8 × 107 parasites were resuspended in 25 μl of lysis buffer (2% SDS, 100 mM Tris-HCl [pH 6.8]) and boiled for 5 min. To enable alkaline phosphatase treatment, the lysate was diluted 20-fold with Tryp wash solution (100 mM NaCl, 3 mM MgCl2 , 20 mM Tris-HCl [pH 7.5]) to reduce the SDS concentration to 0.05% and subsequently reduced to a volume of 100 μl by using an Amicon Ultra 0.5-ml filter device (Millipore). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Chromatin Immunoprecipitation:

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
    Article Snippet: For 3C analysis cells were crosslinked and digested as described for ChIP . .. Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking.

    Plasmid Preparation:

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: The pRNDM plasmid will be provided on request. .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication ( , ), can explain an occurrence of large amounts of double-stranded DNA following a RCR process. .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: A plasmid carrying the gata2 basal promoter linked to the GFP-polyA , which is available from R. M. Grainger, may provide more sensitivity for the co-transgenesis assay since it appears to impart higher levels of transcriptional activity to enhancers without causing background expression on its own. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

    Software:

    Article Title: The association between Alu hypomethylation and severity of type 2 diabetes mellitus
    Article Snippet: Alu PCR products were subjected to COBRA using 2 U of TaqI (Thermo scientific, USA), 2 U of TasI (Thermo scientific, USA), 5x NEB3 buffer (New England Biolabs, USA), and 1 μg/ul bovine serum albumin (BSA) (New England Biolabs, USA) and incubated at 65 °C overnight. .. The cut PCR products were analyzed by 8% acrylamide gel and SYBR stain (Lonza, USA).

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB).

    Electrophoresis:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB).

    Negative Control:

    Article Title: Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia
    Article Snippet: The LINE-1 amplicons (160 bp) were digested with 2 U of Taq I and 2 U of Tas I in NEB3 buffer (New England Biolabs, Ontario, Canada) at 65°C overnight. .. The products were identified by polyacrylamide gel electrophoresis (8% non-denaturing) and stained with SYBR green nucleic acid stain (Sigma-Aldrich, St. Louis, Missouri).

    Affinity Chromatography:

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: For the generation of a polyclonal anti-RPB1 immune serum, the CTD portion of RPB1, comprising the C-terminal 228 amino acids, was expressed with an N-terminal glutathione S -transferase (GST) tag in Escherichia coli , purified by glutathione affinity chromatography, and used as a GST fusion protein to immunize rats according to a previously published protocol ( ). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Agarose Gel Electrophoresis:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB).

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: For the following, generic reagents and equipment are likely to suffice. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge .. The following sections describe how to identify and analyze enhancers in Xenopus , using as an example our analysis of the 5’ flanking region of a paired domain homeobox gene, pax6 ( ).

    Concentration Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: Here we report on the results following this observation and show that a model in which the polymerase shifts from its circular template to its displaced single-stranded DNA, in a similar fashion suggested to sometimes occur in phi29 viral replication ( , ), can explain an occurrence of large amounts of double-stranded DNA following a RCR process. .. We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1
    Article Snippet: To demonstrate that the upper RPB1 band detected in immunoblots with anti-HA or anti-RPB1 antibodies corresponded to phosphorylated RPB1, 8 × 107 parasites were resuspended in 25 μl of lysis buffer (2% SDS, 100 mM Tris-HCl [pH 6.8]) and boiled for 5 min. To enable alkaline phosphatase treatment, the lysate was diluted 20-fold with Tryp wash solution (100 mM NaCl, 3 mM MgCl2 , 20 mM Tris-HCl [pH 7.5]) to reduce the SDS concentration to 0.05% and subsequently reduced to a volume of 100 μl by using an Amicon Ultra 0.5-ml filter device (Millipore). .. The phosphatase reaction was carried out in a volume of 30 μl containing 20 μl of sample, 1× NEB3 buffer, and 10 units of calf intestinal phosphatase (New England BioLabs), and incubation at 37°C for 1 h. To block the phosphatase, 0.5 μl of phosphatase inhibitor cocktail 2 (Sigma) was added to the reaction mixture.

    Marker:

    Article Title: Fitness Analyses of All Possible Point-Mutants for Regions of Genes in Yeast
    Article Snippet: BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) ! .. BsaI restriction endonuclease (New England Biolabs, cat. no.R0535) SphI restriction endonuclease (New Englan Biolabs, cat. no.R0182) MmeI restriction endonuclease (New England Biolabs, cat. no. R0637L) S-adenosyl methionine (SAM; New England Biolabs, cat. no. B9003S) NEB3 buffer (10× with 100× BSA; New England Biolabs, cat. no.B7003) NEB4 buffer (10×; New England Biolabs, cat. no. B7004S) Agarose, PCR grade (Fisher Bioreagents, cat. no. 9012-36-6) Ethidium bromide (Sigma, cat. no. E1510) !

    Gel Extraction:

    Article Title: Comparative genomics-based identification and analysis of cis-regulatory elements
    Article Snippet: ・ QIAquick PCR Purification Kit (Qiagen) ・ QIAquick Gel Extraction kit (Qiagen) ・ Primer pairs for amplification of CNEs: 10 pmol/μL in TE (see Methods for details), stored at −20°C. .. ・ 50 mM MgSO4 (included in a set of the Platinum Pfx DNA polymerase) ・ dNTP mixture, 2.5 mM each (Invitrogen) ・ Bam HI: 10-20 U/μL (New England Biolabs) ・ Xho I: 10-20 U/μL (New England Biolabs) ・ 10× NEB3 buffer (for double-digestion with Bam HI and Xho I, New England Biolabs) ・ 1 × TAE buffer: 40 mM Tris-acetate, 1mM EDTA, pH 8.0 ・ Agarose (molecular biology grade) ・ Ethidium bromide solution: 10 μg/mL (Sigma) ・ TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8.0 ・ 10 mM KCl ・ Ethanol (96-100%, molecular biology grade) ・ 70% Ethanol (prepare by mixing 96-100% ethanol and distilled water) ・ Distilled water ・ Heating block or water bath (for restriction enzyme digestion at 37°C and for melting agarose gel at 50°C) ・ GeneAmp PCR System 2400 (PerkinElmer) or an equivalent standard thermal cycler ・ UV transilluminator (for visualizing ethidium bromide-stained DNA after gel electrophoresis) ・ Microcentrifuge

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    New England Biolabs neb3 buffer
    Neb3 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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