sca1  (New England Biolabs)


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  • 90
    Name:
    ScaI HF
    Description:
    ScaI HF 5 000 units
    Catalog Number:
    r3122l
    Price:
    264
    Size:
    5 000 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs sca1
    ScaI HF
    ScaI HF 5 000 units
    https://www.bioz.com/result/sca1/product/New England Biolabs
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sca1 - by Bioz Stars, 2020-01
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: The Iμ standard was amplified using cDNA template and primers Iμ-5′ 51019 (5′-GCTTGAGTAGTTCTAGTTTCCCCAAACTTAAG-3′) and Iμ-3′ 50615 (5′-GAGTTGGTGGTTGGTCGTACAAGTTG-3′) and cloned into a pGEM-T-easy (Promega) cloning vector. .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′).

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs). .. PCR products were phosphorylated and restricted plasmids dephosphorylated using T4 polynucleotide kinase and Antarctic Phosphatase (New England BioLabs), respectively.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: mRNA preparation and injection optn (ENSDART00000014036.10, Ensembl) and p62 (ENSDART00000140061.2, Ensembl) cDNAs were amplified from 3 dpf WT embryos by PCR (primers in ) and ligated into a vector using the Zero-blunt cloning PCR kit (450245, Invitrogen). .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: Two TOPO clone DNA mixtures were prepared: (i) the four mutant clones were combined at the appropriate ratios to obtain a 10-fold dilution curve and (ii) the mutant mixture described in (i) was diluted into the wild-type background to render a 10−2 total mutant frequency. .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone.

    Centrifugation:

    Article Title: Identification of residues within ligand binding domain 1 (LBD1) of the B. burgdorferi OspC protein required for function in the mammalian environment
    Article Snippet: Cultures were grown at 33°C to mid-log phase in BSK-H medium with 6% rabbit serum (Sigma), collected by centrifugation, washed with cold Dulbecco’s PBS and washed twice with cold EPS buffer (93 g L−1 sucrose, 15% glycerol). .. Fifty μL of the cell suspension was mixed with 20 μg of plasmid linearized with MscI and ScaI-HF (New England Biolabs).

    Amplification:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: Similarly, the VB1-8 standard was amplified using primers VB1-8 -2F (5′-CTGAGCACACAGGACCTCACC-3′) and VB1-8 -2R(5′-GGACTCACCTGAGGAGACTGTG-3′). .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′).

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: The amplification temperatures and elongation times of each PCR were adjusted to the nucleotide sequences of the primers and to the expected size of the PCR product, respectively ( ). .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs).

    Article Title: Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development
    Article Snippet: Paragraph title: RNA isolation and template amplification ... Replicates were pooled and digested with BsrGI and ScaI-HF (NEB) to eliminate contaminating hexamer repeats observed in previous experiments (data not shown).

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Construct:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    SYBR Green Assay:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Incubation:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: After incubation at 30 °C for 30 min, reactions were stopped with addition of 30 μl of stop solution (0.35% SDS, 0.3 mg/ml Proteinase K (Sigma-Aldrich), 400 mm NaCl, 0.3 mg/ml glycogen, and 13 mm EDTA) and incubated for 15 min at 55 °C. .. Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer.

    Article Title: Identification of residues within ligand binding domain 1 (LBD1) of the B. burgdorferi OspC protein required for function in the mammalian environment
    Article Snippet: Fifty μL of the cell suspension was mixed with 20 μg of plasmid linearized with MscI and ScaI-HF (New England Biolabs). .. After 5 min on ice, the cells were electroporated (0.2 cm cuvette, 2.5 kV, 25 μF, 200 Ω), transferred to 10 mL BSK medium with 6% rabbit serum, and incubated overnight at 33 °C.

    Article Title: Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure
    Article Snippet: After the addition of MgCl2 , the samples were incubated on ice for 10 min, centrifuged at 17,000×g for 10 min, and the pellet and supernatant were separated. .. Briefly, the equivalent of 1 pmole nucleosomes were digested in 3.75 µL total volume containing 10 U of ScaI-HF (New England BioLabs), 100 mM KCl, 10 mM Tris pH 7.5, 0.5 mM MgCl2 , 1 mM DTT.

    Stripping Membranes:

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: PCR and restriction enzyme genotyping Red fluorescent F1 progeny (n = 24 total), from successfully injected P0s, were singled to 35 mM NGM dishes and allowed to lay F2 eggs for 2 d. After egg-laying, individual F1s were placed in 7 μl of PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl pH = 8.3, 2.5 mM MgCl2 , 0.45% NP-40, 0.45% Tween-20) containing 1 mg/ml proteinase K in PCR strip tubes. .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.

    Expressing:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: Bacterial recombination assay Efficiencies were measured by co-transforming 75 ng of a pARC8-based expression plasmid (WT, Y324F, R32V, R32M, or 303GVSdup) and an equimolar amount of either pZE2-loxP/loxP or pZE2-ψCore h7q21/ψlox h7q21 into 50 μL vial of OneShot Top10 chemically competent cells. .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: The bicistronic parental plasmid encodes the cell culture-adapted HCV replicon with the ET substitutions and contains the poliovirus IRES in the 5′ end to increase luciferase expression and the EMCV IRES-driven NS3-NS5B HCV coding sequences ( ). .. In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm.

    Touchdown PCR:

    Article Title: Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development
    Article Snippet: Fragments were amplified using FastStart enzyme with 0.5 µM of the same telomeric primer used for reverse transcription through 40 cycles of touchdown PCR (70°–55° for 30 cycles, then 10 cycles at 55°). .. Replicates were pooled and digested with BsrGI and ScaI-HF (NEB) to eliminate contaminating hexamer repeats observed in previous experiments (data not shown).

    Activated Clotting Time Assay:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. DNA was cross-linked to the blot using the autocross-link function on a Stratagene UV Stratalinker 2400, probed with a biotinylated oligo that hybridized to the mispair-containing strand (5′-ATT ATC CCG TAT TGA CGC CGG GCA AGA GCA ACT CGG TCG CCG CAT ACA CT) for 3 h at 55 °C in UltraHyb buffer (Invitrogen), and developed using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

    Hybridization:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. The labeling of probe and hybridization with the membranes were performed according to the manufacturer’s instructions (Amersham Rediprime II labeling system, GE Healthcare).

    Electroporation:

    Article Title: Identification of residues within ligand binding domain 1 (LBD1) of the B. burgdorferi OspC protein required for function in the mammalian environment
    Article Snippet: The resulting plasmids were introduced into B. burgdorferi B31-5A4 (kindly provided by Dr. Jon Skare) by electroporation essentially as previously described with some modifications ( ; ). .. Fifty μL of the cell suspension was mixed with 20 μg of plasmid linearized with MscI and ScaI-HF (New England Biolabs).

    Southern Blot:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: .. Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. About 10 μg digested DNA from each clone was separated on 0.8% agarose gels and transferred to hybond-N+ nylon membrane (GE Healthcare, Piscataway, NJ, USA).

    Ligation:

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs). .. PCR products were phosphorylated and restricted plasmids dephosphorylated using T4 polynucleotide kinase and Antarctic Phosphatase (New England BioLabs), respectively.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106). .. The Luciferase T7 control plasmid (Promega, Cat. No. L4821) was also digested with ScaI HF and PvuI, and treated with thermosensitive alkaline phosphatase (Promega, Cat. No. M9910) to remove the 5′-phosphate groups from the linearized plasmid DNA, thus preventing recircularization during ligation.

    Generated:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. The GFP probe targeted to the GFP transgene was generated by PCR, and the primer sequences are listed in Supplementary Table S1 .

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Imaging:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. Blots were scanned on a Bio-Rad ChemiDoc MP Imaging System, quantitation was performed using Image Lab software, and graphs were made with GraphPad Prism 6.

    Recombination Assay:

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: Paragraph title: Bacterial recombination assay ... The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Sequencing:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure
    Article Snippet: Since the 601 repeats in the 12mer DNA sequence are separated by ScaI restriction enzyme sites, the full occupancy of the 601 sites and the nucleosome positioning could be assessed by ScaI digestion. .. Briefly, the equivalent of 1 pmole nucleosomes were digested in 3.75 µL total volume containing 10 U of ScaI-HF (New England BioLabs), 100 mM KCl, 10 mM Tris pH 7.5, 0.5 mM MgCl2 , 1 mM DTT.

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: The presence of the mutations in the plasmid was verified by DNA sequence analysis. .. In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm.

    Injection:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: Paragraph title: mRNA preparation and injection ... The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain.

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: PCR and restriction enzyme genotyping Red fluorescent F1 progeny (n = 24 total), from successfully injected P0s, were singled to 35 mM NGM dishes and allowed to lay F2 eggs for 2 d. After egg-laying, individual F1s were placed in 7 μl of PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl pH = 8.3, 2.5 mM MgCl2 , 0.45% NP-40, 0.45% Tween-20) containing 1 mg/ml proteinase K in PCR strip tubes. .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.

    Mutagenesis:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Briefly, the 40-μl reactions contained 73 pmol of Msh2–Msh6 (WT or mutant), 54 pmol of PCNA, 41 pmol of RFC-Δ1N, 54 pmol of Mlh1–Pms1, and 100 ng of DNA substrate and were performed in a buffer containing 20 mm HEPES, pH 7.6, 140 mm KCl, 5 mm MgCl2 , 2 mm ATP, 1 mm DTT, 0.2 mg/ml BSA (Roche), 1.2% glycerol (v/v), and 0.5 mm MnSO4 . .. Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: Two TOPO clone DNA mixtures were prepared: (i) the four mutant clones were combined at the appropriate ratios to obtain a 10-fold dilution curve and (ii) the mutant mixture described in (i) was diluted into the wild-type background to render a 10−2 total mutant frequency. .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone.

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: Paragraph title: Construction and characterization of HCV mutant replicons. ... In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm.

    Isolation:

    Article Title: Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development
    Article Snippet: Paragraph title: RNA isolation and template amplification ... Replicates were pooled and digested with BsrGI and ScaI-HF (NEB) to eliminate contaminating hexamer repeats observed in previous experiments (data not shown).

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: The plasmids were isolated using the QIAprep Spin Miniprep kit, eluting into 50 μL10 mM Tris-HCl pH 8. .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Luciferase:

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: The bicistronic parental plasmid encodes the cell culture-adapted HCV replicon with the ET substitutions and contains the poliovirus IRES in the 5′ end to increase luciferase expression and the EMCV IRES-driven NS3-NS5B HCV coding sequences ( ). .. In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106). .. The Luciferase T7 control plasmid (Promega, Cat. No. L4821) was also digested with ScaI HF and PvuI, and treated with thermosensitive alkaline phosphatase (Promega, Cat. No. M9910) to remove the 5′-phosphate groups from the linearized plasmid DNA, thus preventing recircularization during ligation.

    Labeling:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. The labeling of probe and hybridization with the membranes were performed according to the manufacturer’s instructions (Amersham Rediprime II labeling system, GE Healthcare).

    Purification:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: .. Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. About 10 μg digested DNA from each clone was separated on 0.8% agarose gels and transferred to hybond-N+ nylon membrane (GE Healthcare, Piscataway, NJ, USA).

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs).

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: .. Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. DNA was cross-linked to the blot using the autocross-link function on a Stratagene UV Stratalinker 2400, probed with a biotinylated oligo that hybridized to the mispair-containing strand (5′-ATT ATC CCG TAT TGA CGC CGG GCA AGA GCA ACT CGG TCG CCG CAT ACA CT) for 3 h at 55 °C in UltraHyb buffer (Invitrogen), and developed using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The DNA from the overnight LB cultures was purified using the QIAquick Spin Miniprep Kit (Qiagen). .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone.

    Article Title: Identification of residues within ligand binding domain 1 (LBD1) of the B. burgdorferi OspC protein required for function in the mammalian environment
    Article Snippet: The plasmids were propagated in Novablue E. coli cells (Novagen) and purified (HiSpeed Plasmid Midi; Qiagen). .. Fifty μL of the cell suspension was mixed with 20 μg of plasmid linearized with MscI and ScaI-HF (New England Biolabs).

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9). .. The digests quantified on 1% agarose gels following purification using the QIAprep Spin Miniprep kit.

    Article Title: Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure
    Article Snippet: After assembly, MMTV nucleosomes and free MMTV DNA were purified from the 12mer nucleosome arrays using selective MgCl2 precipitation (2–3 mM for WT and Ub-H2A arrays, 4–5 mM for Ub-H2B arrays). .. Briefly, the equivalent of 1 pmole nucleosomes were digested in 3.75 µL total volume containing 10 U of ScaI-HF (New England BioLabs), 100 mM KCl, 10 mM Tris pH 7.5, 0.5 mM MgCl2 , 1 mM DTT.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: .. The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106). .. The Luciferase T7 control plasmid (Promega, Cat. No. L4821) was also digested with ScaI HF and PvuI, and treated with thermosensitive alkaline phosphatase (Promega, Cat. No. M9910) to remove the 5′-phosphate groups from the linearized plasmid DNA, thus preventing recircularization during ligation.

    Polymerase Chain Reaction:

    Article Title: DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
    Article Snippet: Southern blotting Genomic DNA was purified from cells by standard phenol/chloroform extraction and digested with ScaI-HF (New England Biolabs) for 6–8 h, supplemented with 1 mM spermidine (Sigma), 100 μg/ml bovine serum albumin (BSA; Sigma) and 50 μg/ml RNAse A (Millipore). .. The GFP probe targeted to the GFP transgene was generated by PCR, and the primer sequences are listed in Supplementary Table S1 .

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs).

    Article Title: Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development
    Article Snippet: For PCR we used eight replicates per time point to reduce jackpot effects. .. Replicates were pooled and digested with BsrGI and ScaI-HF (NEB) to eliminate contaminating hexamer repeats observed in previous experiments (data not shown).

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: mRNA preparation and injection optn (ENSDART00000014036.10, Ensembl) and p62 (ENSDART00000140061.2, Ensembl) cDNAs were amplified from 3 dpf WT embryos by PCR (primers in ) and ligated into a vector using the Zero-blunt cloning PCR kit (450245, Invitrogen). .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain.

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: One contained wild-type SKOV-3 TP53 exon 5 DNA (named WT), one contained CaOV3 TP53 exon 5 DNA (406 C > T, named MUT1) and three additional clones (MUT2, MUT3 and MUT4) containing distinct TP53 exon 5 mutations (455 C > T, 394 A > G and 431 A > G, respectively), likely introduced by PCR errors. .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: .. The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106). .. The Luciferase T7 control plasmid (Promega, Cat. No. L4821) was also digested with ScaI HF and PvuI, and treated with thermosensitive alkaline phosphatase (Promega, Cat. No. M9910) to remove the 5′-phosphate groups from the linearized plasmid DNA, thus preventing recircularization during ligation.

    Gel Extraction:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106). .. The linear plasmid was gel-purified using QIAquick gel extraction kit (QIAGEN Cat. No. 28706) to remove the undigested plasmid and the excised fragment.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. DNA was cross-linked to the blot using the autocross-link function on a Stratagene UV Stratalinker 2400, probed with a biotinylated oligo that hybridized to the mispair-containing strand (5′-ATT ATC CCG TAT TGA CGC CGG GCA AGA GCA ACT CGG TCG CCG CAT ACA CT) for 3 h at 55 °C in UltraHyb buffer (Invitrogen), and developed using a Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

    Mouse Assay:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: Total cellular RNA was collected from unstimulated (day 0) and stimulated (day 2) B cells from B1-8 mice. .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′).

    Plasmid Preparation:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs). .. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany), and purified plasmids were used to transform electrocompetent A. baumannii NIPH 60 as described previously for Pseudomonas aeruginosa , by using the Gene Pulser II system (Bio-Rad, Munich, Germany).

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. The appropriate bands corresponding to the TP53 exon 5 insert were excised from the gel and purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified via spectrophotometry.

    Article Title: Identification of residues within ligand binding domain 1 (LBD1) of the B. burgdorferi OspC protein required for function in the mammalian environment
    Article Snippet: .. Fifty μL of the cell suspension was mixed with 20 μg of plasmid linearized with MscI and ScaI-HF (New England Biolabs). .. After 5 min on ice, the cells were electroporated (0.2 cm cuvette, 2.5 kV, 25 μF, 200 Ω), transferred to 10 mL BSK medium with 6% rabbit serum, and incubated overnight at 33 °C.

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: Bacterial recombination assay Efficiencies were measured by co-transforming 75 ng of a pARC8-based expression plasmid (WT, Y324F, R32V, R32M, or 303GVSdup) and an equimolar amount of either pZE2-loxP/loxP or pZE2-ψCore h7q21/ψlox h7q21 into 50 μL vial of OneShot Top10 chemically competent cells. .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: The presence of the mutations in the plasmid was verified by DNA sequence analysis. .. In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: Paragraph title: Restriction digestion of PCR product and vector. ... The PCR products were digested with ScaI HF and PvuI restriction endonucleases (NEB, Cat. No. R3122 and R0150) according to the manufacturer’s protocol and was purified using QIAquick PCR purification kit (QIAGEN, Cat. No. 28106).

    Software:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Reactions were performed using a 7900HT real-time instrument (Applied Biosystems) and data analyzed using SDS 2.3 software (Applied Biosystems).

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. Blots were scanned on a Bio-Rad ChemiDoc MP Imaging System, quantitation was performed using Image Lab software, and graphs were made with GraphPad Prism 6.

    Real-time Polymerase Chain Reaction:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Agarose Gel Electrophoresis:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′). .. Each primer set was examined for > 90% amplification efficiency and for the lack of secondary products.

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. DNA representing 3.6 million different barcoded CypherSeq vectors (as estimated by bacterial colony counts) containing the N701 Illumina index (hereafter named ‘N701-CypherSeq’) was digested with SmaI (New England BioLabs) and treated with Antarctic phosphatase (New England BioLabs) before electrophoresis on a 1.5% low melting-point agarose gel containing 1× SYBR Safe (Life Technologies).

    In Vitro:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: .. In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm. .. In vitro -transcribed RNA was electroporated into Huh-cure cells (obtained from Ralf Bartenschlager) suspended to a concentration of 1 × 107 cells/ml in cytomix medium (120 mM KCl, 0.15 mM CaCl2 , 10 mM K2 HPO4 -KH2 PO4 , 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2 , and freshly added 2 mM ATP and 5 mM glutathione; pH 7.6).

    Electrophoresis:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. The appropriate bands corresponding to the TP53 exon 5 insert were excised from the gel and purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified via spectrophotometry.

    Ethanol Precipitation:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: The DNA products were purified by phenol extraction and ethanol precipitation. .. Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer.

    Quantitation Assay:

    Article Title: The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
    Article Snippet: Purified DNA was digested with 10 units of ScaI-HF (New England Biolabs), and 10 ng were run on 1% denaturing agarose gels at 25 V for 3 h and transferred to an Amersham Biosciences Hybond-N+ membrane (GE Healthcare) using the capillary transfer method with 20× SSC buffer. .. Blots were scanned on a Bio-Rad ChemiDoc MP Imaging System, quantitation was performed using Image Lab software, and graphs were made with GraphPad Prism 6.

    Spectrophotometry:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. The appropriate bands corresponding to the TP53 exon 5 insert were excised from the gel and purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified via spectrophotometry.

    Produced:

    Article Title: Immunoglobulin switch ? sequence causes RNA polymerase II accumulation and reduces dA hypermutation
    Article Snippet: Complementary DNA (cDNA) was produced using 0.5 µg and 1.0 µg RNA and the iScript cDNA synthesis kit (Bio-Rad Laboratories). .. For the standard curve, plasmid standards were linearized with ScaI-HF (New England Biolabs, Inc.) and purified by agarose gel electrophoresis and gel extraction (QIAGEN). qPCR was performed using IQ SYBR green supermix (Bio-Rad Laboratories) with primers Iμ-5′ 50827 (5′-CCAATACCCGAAGCATTTACAGTGAC-3′) and Iμ-3′ 50726 (5′-GTGAAGCCGTTTTGACCAGAATGTC-3′) and with primers VB1-8 -3F (5′-GACGAGGCCTTGAGTGGATTG-3′) and VB1-8 -3R (5′-CATGTAGGCTGTGCTGGAGG-3′).

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Concentration Assay:

    Article Title: In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen, a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122
    Article Snippet: In vitro -transcribed replicon RNA was prepared by runoff transcription of ScaI -HF (New England BioLabs, Ipswich, MA) restriction enzyme-digested plasmids using MegaScript T7 RNA reagents (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended procedure and quantified by absorbance at 260 nm. .. In vitro -transcribed RNA was electroporated into Huh-cure cells (obtained from Ralf Bartenschlager) suspended to a concentration of 1 × 107 cells/ml in cytomix medium (120 mM KCl, 0.15 mM CaCl2 , 10 mM K2 HPO4 -KH2 PO4 , 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2 , and freshly added 2 mM ATP and 5 mM glutathione; pH 7.6).

    Lysis:

    Article Title: The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii
    Article Snippet: A. baumannii genomic DNA was extracted by heat lysis. .. DNA restriction was performed using the endonucleases EcoRI-HF, NcoI-HF, and ScaI-HF (New England BioLabs), and cloning was performed according to the manufacturer's recommendations using the Quick Ligation kit (New England BioLabs).

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: PCR and restriction enzyme genotyping Red fluorescent F1 progeny (n = 24 total), from successfully injected P0s, were singled to 35 mM NGM dishes and allowed to lay F2 eggs for 2 d. After egg-laying, individual F1s were placed in 7 μl of PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl pH = 8.3, 2.5 mM MgCl2 , 0.45% NP-40, 0.45% Tween-20) containing 1 mg/ml proteinase K in PCR strip tubes. .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.

    Staining:

    Article Title: Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure
    Article Snippet: The reconstituted arrays were analyzed on native 1% agarose/2% polyacrylamide (APAGE) gels stained with Sybr Gold nucleic acid gel stain (Life Technologies). .. Briefly, the equivalent of 1 pmole nucleosomes were digested in 3.75 µL total volume containing 10 U of ScaI-HF (New England BioLabs), 100 mM KCl, 10 mM Tris pH 7.5, 0.5 mM MgCl2 , 1 mM DTT.

    Clear Native PAGE:

    Article Title: Multivalency governs HP1α association dynamics with the silent chromatin state
    Article Snippet: .. 0.5–2 pmol of chromatin arrays were digested with ScaI-HF (NEB) in 10 μl for 6 h. Quality of the reconstitution was assessed by native PAGE ( ). ..

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    New England Biolabs sca1
    Sca1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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