bamhi restriction enzyme  (New England Biolabs)


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    Name:
    BamHI
    Description:
    BamHI 50 000 units
    Catalog Number:
    r0136l
    Price:
    244
    Size:
    50 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs bamhi restriction enzyme
    BamHI
    BamHI 50 000 units
    https://www.bioz.com/result/bamhi restriction enzyme/product/New England Biolabs
    Average 90 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    bamhi restriction enzyme - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Evidence Suggesting Absence of Mitochondrial DNA Methylation"

    Article Title: Evidence Suggesting Absence of Mitochondrial DNA Methylation

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2017.00166

    BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.
    Figure Legend Snippet: BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Techniques Used: Methylation Sequencing, DNA Methylation Assay, Methylation

    Related Articles

    Clone Assay:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
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    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 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.

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    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma
    Article Snippet: Transcription terminal signal was cloned from the BGH polyA of pcDNA3.1 (+) (Life Technologies, V790-20). .. Every two neighbouring fragments were ligated by Bam HI/Bgl II (New England Biolabs, Ipswich, MA, USA; R0136/R0144) cohesive ends [ ].

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
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    Article Title: Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency
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    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
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    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
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    Amplification:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
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    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
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    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
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    Article Title: Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis
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    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
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    Mass Spectrometry:

    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
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    Synthesized:

    Article Title: Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
    Article Snippet: .. Free-living adult worms Genomic DNA extraction Gentra Puregene Tissue Kit (QIAGEN), including: Cell lysis solution (catalog number: 8304295) Protein precipitation solution (catalog number: 8273807) DNA hydration solution (catalog number: 8274043) Enzymes for digestion and treatment Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs) Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05) and Exonuclease I (New England Biolabs, catalog number: M0293S) Ligation reagents 10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S) PCR reagents 5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S) Oligonucleotides and primers The oligonucleotides detailed in must be synthesized .. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf) Gel electrophoresis Gel documentation (FOTODYNE, model: FOTO/Analyst FX)

    Cytometry:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: The cells were washed and resuspended in MACS buffer and analyzed on a FACSCalibur flow cytometer as described above. .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Construct:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The novel triple fusion reporter plasmid, DsRedm-fl-ttksr39 (p3H), was constructed essentially as described by Ray et al. [ , ]. .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. 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. .. 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.

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma
    Article Snippet: Another plasmid, pDC312/AFP, an empty vector without exogenous genes was constructed as control as schematically described in Figure B. .. Every two neighbouring fragments were ligated by Bam HI/Bgl II (New England Biolabs, Ipswich, MA, USA; R0136/R0144) cohesive ends [ ].

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Real-time Polymerase Chain Reaction:

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops. .. After washing and treatment with Proteinase K (20 ng, 15 min at 45°C) DNA was purified and analyzed by qPCR.

    Incubation:

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops. .. 10 μg of fragmented DNA were incubated overnight with 10 μg S9.6 antibody in 0.1 ml IP buffer (20 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 10 mM MgCl2 , 0.5% Triton X-100, 1mM dithiothreitol) and RNA:DNA hybrids were bound to protein A/G agarose (Sigma).

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: For screening of individual clones, 1×107 induced yeast cells were incubated on a shaking incubator with 33 µg/µl Alexa-488 conjugated nymph-immune rabbit IgG in MACS buffer for 45 minutes at room temperature. .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Expressing:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: Briefly, the monomeric DsRed expression plasmid, pDsRed-Monomer -C1 , driven by CMV enhancer/promoter, was purchased from Clontech (BD science, Inc., USA). .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector.

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: Paragraph title: Selection of yeast cells expressing immunogenic I. scapularis nymph salivary proteins ... Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. For the PAK4 knockout, the PAK4-specific siRNA (siRNA-PAK4) or siRNA-Con (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 25 or 50 nM were transfected with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) into the Hela or Caski cells to abrogate the HIWI expression.

    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: Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis
    Article Snippet: .. The amplified fragment was cloned into pGEM T-Easy Vector System (Promega) and the resulting clone was verified by sequencing. pGEM T-Easy-capzb plasmid was digested with BamH I and Xho I (New England Biolabs, Ipswich, MA, USA) and subcloned into pCS2+ expression vector for subsequent capped mRNA synthesis. .. Spe I-linearized pGEM T-Easy- capzb and Kpn I-linearized pCS2+ - capzb (described above) were used to synthesize DIG-labeled anti-sense and sense riboprobes, using the T7 mMESSAGE mMACHINE and the SP6 mMESSAGE mMACHINE RNA Synthesis Kit (Ambion, Foster City, CA, USA), respectively, according to the manufacturer's instructions.

    Article Title: The ?33-35 Mutant α-Domain Containing β-Domain-Like M3S9 Cluster Exhibits the Function of α-Domain with M4S11 Cluster in Human Growth Inhibitory Factor
    Article Snippet: Reagents Fusion expression vector pGEX-4T-2, Escherichia coli strain BL21, glutathione Sepharose 4B, Superdex-75, and Sephadex G-25 were purchased from Amersham Pharmacia Biotech. .. The (deoxy-ribonucleoside triphosphate) dNTP, T4 DNA ligase, and restriction enzymes, BamH I and EcoR I , were purchased from New England Biolabs.

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma
    Article Snippet: The tandem repeats of artificial miRNAs were ligated into adenoviral shuttle vector pDC312 under recombinant AFP enhancer/promoter to form the artificial miRNAs expression cassette AFP-amiR, followed by the AFP-Casp expression cassette in the same orientation, as schematically depicted in Figure B. .. Every two neighbouring fragments were ligated by Bam HI/Bgl II (New England Biolabs, Ipswich, MA, USA; R0136/R0144) cohesive ends [ ].

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). .. Expression plasmids were grown in Luria-Bertani (LB) medium containing 30 μg/mL kanamycin at 310 K until the OD600 reached 0.65–1.0.

    Modification:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: Cell culture and treatment Human cervical cancer Hela and Caski cells were purchased from American Type Culture Collection (ATCC) (Rockville, MD, USA) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (for Hela cells) or RPMI-1640 medium (for Caski cells), which was supplemented with 10 % fetal bovine serum (FBS) (GIBCO, Rockville, MD, USA), with 100 U/mL penicillin and 100 μg/mL streptomycin (CSPC, Shijiazhuang, China). .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4.

    Transformation Assay:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: Plasmid DNA was isolated from individual positive clones using the ZymoprepTM II Yeast Plasmid Miniprep kit (Zymo research, Orange, CA), transformed into E. coli DH5α (Invitrogen, CA) and plated on LB plates containing 100 µg/ml ampicillin. .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: The plasmid was transformed into One Shot Chemically Competent E. coli (LifeTechnologies), purified and sequenced by SRD (Scientific Research and Development, Bad Homburg, Germany). .. For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany).

    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
    Article Snippet: .. Vector Construction and Genetic Transformation The open reading frames of GhFPF1 (GenBank Accession No. KC832319) was cloned into the binary vector pBI121 using an In-Fusions ™ Advantage PCR Cloning Kit (Clontech, USA) via BamH I and Sac I sites (New England BioLabs, USA). .. The recombinant plasmid containing CaMV35S::GhFPF1 was introduced into Agrobacterium tumefaciens (strain LBA4404) and then transformed into Arabidopsis (Columbia-0 ecotype) according to the method of floral dip .

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). .. E. coli BL 21 (DE 3 )-RIL (TaKaRa) cultures were transformed into vectors involving aforementioned recombinant plasmid.

    Over Expression:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: For the cisplatin (Sigma-Aldrich, St. Louis, MO, USA) treatment, 85 % or higher confluent Hela or Caski cells were updated with DMEM or RPMI-1640 medium supplemented with 2 % FBS, and with 5 μM (for Hela cells) or 10 μM (for CaSki cells) cisplatin for 12, 24 or 48 h; For the PAK4 overexpression, the open reading frame (ORF) of PAK4 (NM_005884.3) was amplified by PCR with Phusion polymerase (New England Biolabs, Ipswich, MA, USA) and with the primers (Forward primer: 5′-ATG TTT GGG AAG AGG AAG AAG C-3′ and Reverse primer: 5′-TCA TCT GGT GCG GTT CTG GCG-3′). .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4.

    Countercurrent Chromatography:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Flow Cytometry:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: The cells were washed and resuspended in MACS buffer and analyzed on a FACSCalibur flow cytometer as described above. .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Ligation:

    Article Title: Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
    Article Snippet: .. Free-living adult worms Genomic DNA extraction Gentra Puregene Tissue Kit (QIAGEN), including: Cell lysis solution (catalog number: 8304295) Protein precipitation solution (catalog number: 8273807) DNA hydration solution (catalog number: 8274043) Enzymes for digestion and treatment Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs) Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05) and Exonuclease I (New England Biolabs, catalog number: M0293S) Ligation reagents 10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S) PCR reagents 5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S) Oligonucleotides and primers The oligonucleotides detailed in must be synthesized .. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf) Gel electrophoresis Gel documentation (FOTODYNE, model: FOTO/Analyst FX)

    Cell Culture:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: Paragraph title: Cell culture and treatment ... The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4.

    Article Title: The ?33-35 Mutant α-Domain Containing β-Domain-Like M3S9 Cluster Exhibits the Function of α-Domain with M4S11 Cluster in Human Growth Inhibitory Factor
    Article Snippet: The (deoxy-ribonucleoside triphosphate) dNTP, T4 DNA ligase, and restriction enzymes, BamH I and EcoR I , were purchased from New England Biolabs. .. Pfu DNA polymerase, cell culture reagents, isopropyl β -D-thiogalactoside (IPTG), and Triton-100 were purchased from Sangon (Shanghai, China).

    Generated:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: 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. .. 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.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: Cellular Localization of SOAT mRNA by in situ Hybridization (ISH) Digoxigenin (DIG)-labeled cRNA probes were generated as described previously . .. For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany).

    Polymerase Chain Reaction:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: Briefly ~400 nt long sense strand of each transcript was prepared by PCR using mouse hippocampus cDNA as a template and transcript specific PCR primers (Table ) and ligated to pCRII-TOPO Vector with dual promoters T7 and SP6 (Invitrogen, Cat. .. The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes.

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: For the cisplatin (Sigma-Aldrich, St. Louis, MO, USA) treatment, 85 % or higher confluent Hela or Caski cells were updated with DMEM or RPMI-1640 medium supplemented with 2 % FBS, and with 5 μM (for Hela cells) or 10 μM (for CaSki cells) cisplatin for 12, 24 or 48 h; For the PAK4 overexpression, the open reading frame (ORF) of PAK4 (NM_005884.3) was amplified by PCR with Phusion polymerase (New England Biolabs, Ipswich, MA, USA) and with the primers (Forward primer: 5′-ATG TTT GGG AAG AGG AAG AAG C-3′ and Reverse primer: 5′-TCA TCT GGT GCG GTT CTG GCG-3′). .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. 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. .. 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.

    Article Title: Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis
    Article Snippet: The full-length 822 bp coding region of capzb (NCBI Reference Sequence: , ) was PCR amplified from total cDNA (described above) using forward primer 5′-GAAGCAGGATCCATGAATGAGC-3′ and reverse primer 5′-GTATTCTCGAGCTAGCTCTGC-3′. .. The amplified fragment was cloned into pGEM T-Easy Vector System (Promega) and the resulting clone was verified by sequencing. pGEM T-Easy-capzb plasmid was digested with BamH I and Xho I (New England Biolabs, Ipswich, MA, USA) and subcloned into pCS2+ expression vector for subsequent capped mRNA synthesis.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: Primer pairs for PCR reaction were obtained from Eurofins MWG Operon ( ). .. For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany).

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Article Title: Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency
    Article Snippet: .. PCR product was cloned into pCDNA3 (Invitrogen Life Tech) with BamH I and Xho I (New England BioLabs, R0146S) restriction enzyme sites and sequenced. .. Ewsr1 MEFs and NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (Hyclone, SH30243.01) supplemented with 10% fetal bovine serum (Hyclone, SH30071.03), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen Life Tech, 15070063).

    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
    Article Snippet: .. Vector Construction and Genetic Transformation The open reading frames of GhFPF1 (GenBank Accession No. KC832319) was cloned into the binary vector pBI121 using an In-Fusions ™ Advantage PCR Cloning Kit (Clontech, USA) via BamH I and Sac I sites (New England BioLabs, USA). .. The recombinant plasmid containing CaMV35S::GhFPF1 was introduced into Agrobacterium tumefaciens (strain LBA4404) and then transformed into Arabidopsis (Columbia-0 ecotype) according to the method of floral dip .

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: In order to obtain the generation of full-length viral cDNA sequence, TMV-RNA was reverse transcribed using primercDNA (Table ) in 50 mmol/L Tris at pH 8.0, 8.0 mmol/L magnesium chloride, 75 mmol/L potassium chloride, 10 mmol/L DL-Dithiothreitol, 1.0 mmol/L dNTPs, 0.5 unit/μL AMV reverse transcriptase (TaKaRa), and 1.0 unit/μL RNase inhibitor (TaKaRa) for 1.5 hr at 315 K. Using the generation of full-length viral cDNA sequence and the corresponding primers by the same method, TR-His-TMV-CP19 , WT-GST-TMV-CP32 , WT-His-TMV-CP12 , TR-His-TMV-CP62 , and TR-His-TMV-CP68 sequences were amplified by PCR technology (Table ). .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    Article Title: Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
    Article Snippet: .. Free-living adult worms Genomic DNA extraction Gentra Puregene Tissue Kit (QIAGEN), including: Cell lysis solution (catalog number: 8304295) Protein precipitation solution (catalog number: 8273807) DNA hydration solution (catalog number: 8274043) Enzymes for digestion and treatment Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs) Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05) and Exonuclease I (New England Biolabs, catalog number: M0293S) Ligation reagents 10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S) PCR reagents 5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S) Oligonucleotides and primers The oligonucleotides detailed in must be synthesized .. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf) Gel electrophoresis Gel documentation (FOTODYNE, model: FOTO/Analyst FX)

    Sequencing:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University). .. Production of recombinant salivary proteins p8, p19 and p23 cDNAs were cloned in frame into the pMT/Bip/V5-HisA plasmid containing a His tag, V5 epitope, and a blasticidin resistance gene (Invitrogen, CA), and validated by sequencing.

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. Hela or Caski cells with more than 85 % confluence were transfected with the recombinant pcDNA3.1(+)-PAK4 or pcDNA3.1(+)-Con plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    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: Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis
    Article Snippet: .. The amplified fragment was cloned into pGEM T-Easy Vector System (Promega) and the resulting clone was verified by sequencing. pGEM T-Easy-capzb plasmid was digested with BamH I and Xho I (New England Biolabs, Ipswich, MA, USA) and subcloned into pCS2+ expression vector for subsequent capped mRNA synthesis. .. Spe I-linearized pGEM T-Easy- capzb and Kpn I-linearized pCS2+ - capzb (described above) were used to synthesize DIG-labeled anti-sense and sense riboprobes, using the T7 mMESSAGE mMACHINE and the SP6 mMESSAGE mMACHINE RNA Synthesis Kit (Ambion, Foster City, CA, USA), respectively, according to the manufacturer's instructions.

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: In order to obtain the generation of full-length viral cDNA sequence, TMV-RNA was reverse transcribed using primercDNA (Table ) in 50 mmol/L Tris at pH 8.0, 8.0 mmol/L magnesium chloride, 75 mmol/L potassium chloride, 10 mmol/L DL-Dithiothreitol, 1.0 mmol/L dNTPs, 0.5 unit/μL AMV reverse transcriptase (TaKaRa), and 1.0 unit/μL RNase inhibitor (TaKaRa) for 1.5 hr at 315 K. Using the generation of full-length viral cDNA sequence and the corresponding primers by the same method, TR-His-TMV-CP19 , WT-GST-TMV-CP32 , WT-His-TMV-CP12 , TR-His-TMV-CP62 , and TR-His-TMV-CP68 sequences were amplified by PCR technology (Table ). .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    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 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.

    Recombinant:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. Hela or Caski cells with more than 85 % confluence were transfected with the recombinant pcDNA3.1(+)-PAK4 or pcDNA3.1(+)-Con plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma
    Article Snippet: The tandem repeats of artificial miRNAs were ligated into adenoviral shuttle vector pDC312 under recombinant AFP enhancer/promoter to form the artificial miRNAs expression cassette AFP-amiR, followed by the AFP-Casp expression cassette in the same orientation, as schematically depicted in Figure B. .. Every two neighbouring fragments were ligated by Bam HI/Bgl II (New England Biolabs, Ipswich, MA, USA; R0136/R0144) cohesive ends [ ].

    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
    Article Snippet: Vector Construction and Genetic Transformation The open reading frames of GhFPF1 (GenBank Accession No. KC832319) was cloned into the binary vector pBI121 using an In-Fusions ™ Advantage PCR Cloning Kit (Clontech, USA) via BamH I and Sac I sites (New England BioLabs, USA). .. The recombinant plasmid containing CaMV35S::GhFPF1 was introduced into Agrobacterium tumefaciens (strain LBA4404) and then transformed into Arabidopsis (Columbia-0 ecotype) according to the method of floral dip .

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). .. E. coli BL 21 (DE 3 )-RIL (TaKaRa) cultures were transformed into vectors involving aforementioned recombinant plasmid.

    Cellular Antioxidant Activity Assay:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The fl gene from the pGL3 basic plasmid (Promega Corporation, Madison, WI, USA) was amplified by PCR using the same 5′-end primer FLUCUp-EcoRI 5′-AGC ATC GAA TTC TGA GGA CGC CAA AAA CAT AAA G-3′, the 3′end primer FLUCDn-SalI 5′-CTA GTA GTC GAC AGC AAT CTT TCC GCC CTT CT-3′, and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Magnetic Cell Separation:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: The cells were washed and resuspended in MACS buffer and analyzed on a FACSCalibur flow cytometer as described above. .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    DNA Extraction:

    Article Title: Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
    Article Snippet: .. Free-living adult worms Genomic DNA extraction Gentra Puregene Tissue Kit (QIAGEN), including: Cell lysis solution (catalog number: 8304295) Protein precipitation solution (catalog number: 8273807) DNA hydration solution (catalog number: 8274043) Enzymes for digestion and treatment Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs) Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05) and Exonuclease I (New England Biolabs, catalog number: M0293S) Ligation reagents 10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S) PCR reagents 5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S) Oligonucleotides and primers The oligonucleotides detailed in must be synthesized .. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf) Gel electrophoresis Gel documentation (FOTODYNE, model: FOTO/Analyst FX)

    In Vivo:

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops. .. To measure 5mC oxidation products in R-loops in vivo , abdomen and heads of 15.5 days mouse embryos were lysed in 500 μl lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl2 , 0.5% Triton X-100, 1mM dithiothreitol).

    Isolation:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University). .. Production of recombinant salivary proteins p8, p19 and p23 cDNAs were cloned in frame into the pMT/Bip/V5-HisA plasmid containing a His tag, V5 epitope, and a blasticidin resistance gene (Invitrogen, CA), and validated by sequencing.

    Transfection:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. Hela or Caski cells with more than 85 % confluence were transfected with the recombinant pcDNA3.1(+)-PAK4 or pcDNA3.1(+)-Con plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Labeling:

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: In situ hybridization DIG labeled ribo probes complementary to selected lncRNAs for in situ hybridization were prepared by in vitro transcription of cDNA templates by using T7 RNA polymerase as described earlier (Kadakkuzha et al., ). .. The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany). .. Subsequently, in vitro transcription of plasmid DNA in cRNA was performed using 10×RNA-DIG Labeling Mix (Boehringer Mannheim, Mannheim, Germany) and RNA polymerases T7 and SP6 (Promega, Heidelberg, Germany).

    Purification:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops. .. After washing and treatment with Proteinase K (20 ng, 15 min at 45°C) DNA was purified and analyzed by qPCR.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. 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. .. 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.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: The plasmid was transformed into One Shot Chemically Competent E. coli (LifeTechnologies), purified and sequenced by SRD (Scientific Research and Development, Bad Homburg, Germany). .. For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany).

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: The dsDNA of correct length was purified and identified by 1% agarose gel electrophoresis. .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Upland Cotton Gene GhFPF1 Confers Promotion of Flowering Time and Shade-Avoidance Responses in Arabidopsis thaliana
    Article Snippet: Vector Construction and Genetic Transformation The open reading frames of GhFPF1 (GenBank Accession No. KC832319) was cloned into the binary vector pBI121 using an In-Fusions ™ Advantage PCR Cloning Kit (Clontech, USA) via BamH I and Sac I sites (New England BioLabs, USA). .. To confirm that GhFPF1 had been integrated into the Arabidopsis genome, RT-PCR was performed with the 35S promoter primer (forward) and the GhFPF1 -specific primer (reverse).

    Blocking Assay:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. Phosphoinositide 3-kinase/ RAC-alpha serine/threonine-protein kinase (PI3K/Akt) inhibitor, LY294002 (Thermo Scientific, Rockford, IL, USA) was utilized with 10 or 20 nM (Hela cells) or with 15 or 30 nM (Caski cells) to block the PI3K/Akt pathway.

    Lysis:

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops. .. To measure 5mC oxidation products in R-loops in vivo , abdomen and heads of 15.5 days mouse embryos were lysed in 500 μl lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl2 , 0.5% Triton X-100, 1mM dithiothreitol).

    Article Title: Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
    Article Snippet: .. Free-living adult worms Genomic DNA extraction Gentra Puregene Tissue Kit (QIAGEN), including: Cell lysis solution (catalog number: 8304295) Protein precipitation solution (catalog number: 8273807) DNA hydration solution (catalog number: 8274043) Enzymes for digestion and treatment Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs) Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05) and Exonuclease I (New England Biolabs, catalog number: M0293S) Ligation reagents 10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S) PCR reagents 5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S) Oligonucleotides and primers The oligonucleotides detailed in must be synthesized .. PCR Thermal Cyclers (Mastercycler Personal, Eppendorf) Gel electrophoresis Gel documentation (FOTODYNE, model: FOTO/Analyst FX)

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. Hela or Caski cells with more than 85 % confluence were transfected with the recombinant pcDNA3.1(+)-PAK4 or pcDNA3.1(+)-Con plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    In Situ Hybridization:

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: Paragraph title: In situ hybridization ... The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: Paragraph title: Cellular Localization of SOAT mRNA by in situ Hybridization (ISH) ... For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany).

    Plasmid Preparation:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: .. The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes. .. A small aliquot (2 μl) was run on 1.5% agarose gel to confirm the integrity of RNA probes and stored at −80°C until used.

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: .. Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University). .. Production of recombinant salivary proteins p8, p19 and p23 cDNAs were cloned in frame into the pMT/Bip/V5-HisA plasmid containing a His tag, V5 epitope, and a blasticidin resistance gene (Invitrogen, CA), and validated by sequencing.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. 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. .. 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.

    Article Title: Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis
    Article Snippet: .. The amplified fragment was cloned into pGEM T-Easy Vector System (Promega) and the resulting clone was verified by sequencing. pGEM T-Easy-capzb plasmid was digested with BamH I and Xho I (New England Biolabs, Ipswich, MA, USA) and subcloned into pCS2+ expression vector for subsequent capped mRNA synthesis. .. Spe I-linearized pGEM T-Easy- capzb and Kpn I-linearized pCS2+ - capzb (described above) were used to synthesize DIG-labeled anti-sense and sense riboprobes, using the T7 mMESSAGE mMACHINE and the SP6 mMESSAGE mMACHINE RNA Synthesis Kit (Ambion, Foster City, CA, USA), respectively, according to the manufacturer's instructions.

    Article Title: The ?33-35 Mutant α-Domain Containing β-Domain-Like M3S9 Cluster Exhibits the Function of α-Domain with M4S11 Cluster in Human Growth Inhibitory Factor
    Article Snippet: Reagents Fusion expression vector pGEX-4T-2, Escherichia coli strain BL21, glutathione Sepharose 4B, Superdex-75, and Sephadex G-25 were purchased from Amersham Pharmacia Biotech. .. The (deoxy-ribonucleoside triphosphate) dNTP, T4 DNA ligase, and restriction enzymes, BamH I and EcoR I , were purchased from New England Biolabs.

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma
    Article Snippet: Another plasmid, pDC312/AFP, an empty vector without exogenous genes was constructed as control as schematically described in Figure B. .. Every two neighbouring fragments were ligated by Bam HI/Bgl II (New England Biolabs, Ipswich, MA, USA; R0136/R0144) cohesive ends [ ].

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: .. For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany). .. Subsequently, in vitro transcription of plasmid DNA in cRNA was performed using 10×RNA-DIG Labeling Mix (Boehringer Mannheim, Mannheim, Germany) and RNA polymerases T7 and SP6 (Promega, Heidelberg, Germany).

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Article Title: Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency
    Article Snippet: Paragraph title: Plasmid construction ... PCR product was cloned into pCDNA3 (Invitrogen Life Tech) with BamH I and Xho I (New England BioLabs, R0146S) restriction enzyme sites and sequenced.

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    Selection:

    Article Title: Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display
    Article Snippet: Paragraph title: Selection of yeast cells expressing immunogenic I. scapularis nymph salivary proteins ... Plasmid DNA was then isolated from bacterial colonies using the Plasmid Miniprep kit (Qiagen, CA), digested with Xho I and BamH I (New England Biolabs, MA) to assess insert sizes and unique clones sent for sequencing (Keck Facility, Yale University).

    Agarose Gel Electrophoresis:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes. .. A small aliquot (2 μl) was run on 1.5% agarose gel to confirm the integrity of RNA probes and stored at −80°C until used.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct.

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: The dsDNA of correct length was purified and identified by 1% agarose gel electrophoresis. .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    In Vitro:

    Article Title: Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations
    Article Snippet: In situ hybridization DIG labeled ribo probes complementary to selected lncRNAs for in situ hybridization were prepared by in vitro transcription of cDNA templates by using T7 RNA polymerase as described earlier (Kadakkuzha et al., ). .. The Vector with the sense lncRNA DNA in the 5–3′ direction was linearized with BamH I (New England Biolab) for transcription using T7 RNA polymerase to generate antisense probes.

    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: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: For the synthesis of cRNA probes, the plasmid was digested by using restriction enzymes Not I or BamH I (NEB, Schwalbach, Germany). .. Subsequently, in vitro transcription of plasmid DNA in cRNA was performed using 10×RNA-DIG Labeling Mix (Boehringer Mannheim, Mannheim, Germany) and RNA polymerases T7 and SP6 (Promega, Heidelberg, Germany).

    Knock-Out:

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4. .. For the PAK4 knockout, the PAK4-specific siRNA (siRNA-PAK4) or siRNA-Con (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 25 or 50 nM were transfected with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) into the Hela or Caski cells to abrogate the HIWI expression.

    Produced:

    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.

    Immunoprecipitation:

    Article Title: GADD45A binds R-loops and recruits TET1 to CpG island promoters
    Article Snippet: Paragraph title: RNA:DNA immunoprecipitation (DRIP) ... In brief, 20 μg DNA were treated with 10 ng/μl of RNase A for 5 min at RT, recovered by PCI extraction and digested with 4 U EcoR I, EcoR V, Xba I, BamH I and Ssp I (NEB) at 37°C for 3h or treated with RNase H1 (0.2 U/μl) for target validation of R-loops.

    CTG Assay:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway
    Article Snippet: For the cisplatin (Sigma-Aldrich, St. Louis, MO, USA) treatment, 85 % or higher confluent Hela or Caski cells were updated with DMEM or RPMI-1640 medium supplemented with 2 % FBS, and with 5 μM (for Hela cells) or 10 μM (for CaSki cells) cisplatin for 12, 24 or 48 h; For the PAK4 overexpression, the open reading frame (ORF) of PAK4 (NM_005884.3) was amplified by PCR with Phusion polymerase (New England Biolabs, Ipswich, MA, USA) and with the primers (Forward primer: 5′-ATG TTT GGG AAG AGG AAG AAG C-3′ and Reverse primer: 5′-TCA TCT GGT GCG GTT CTG GCG-3′). .. The ORF sequence was then cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA), with Hind III (New England BioLabs, Beverly, MA, USA) and BamH I (New England BioLabs, Beverly, MA, USA) as restriction enzymes, and with the chloramphenicol acetyltransferase (CAT) as a control for PAK4.

    Gel Extraction:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. 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. .. 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.

    Article Title: The ?33-35 Mutant α-Domain Containing β-Domain-Like M3S9 Cluster Exhibits the Function of α-Domain with M4S11 Cluster in Human Growth Inhibitory Factor
    Article Snippet: The (deoxy-ribonucleoside triphosphate) dNTP, T4 DNA ligase, and restriction enzymes, BamH I and EcoR I , were purchased from New England Biolabs. .. The DNA gel extraction kit was purchased from Qiagen.

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    New England Biolabs bamhi restriction enzyme
    <t>BamHI</t> digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested <t>DNA</t> methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.
    Bamhi Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Journal: Frontiers in Genetics

    Article Title: Evidence Suggesting Absence of Mitochondrial DNA Methylation

    doi: 10.3389/fgene.2017.00166

    Figure Lengend Snippet: BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Article Snippet: Restriction Enzyme Treatment Genomic DNA was either untreated or treated with BamHI restriction enzyme (New England Biolabs, cat # R0136) to obtain circular or linearized mtDNA.

    Techniques: Methylation Sequencing, DNA Methylation Assay, Methylation