neb 5 alpha f  (New England Biolabs)


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    Name:
    NEB 5 alpha F Iq Competent E coli High Efficiency
    Description:
    NEB 5 alpha F Iq Competent E coli High Efficiency 20x0 05 ml
    Catalog Number:
    c2992h
    Price:
    198
    Size:
    1 ml
    Category:
    Competent Bacteria
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    Structured Review

    New England Biolabs neb 5 alpha f
    NEB 5 alpha F Iq Competent E coli High Efficiency
    NEB 5 alpha F Iq Competent E coli High Efficiency 20x0 05 ml
    https://www.bioz.com/result/neb 5 alpha f/product/New England Biolabs
    Average 95 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    neb 5 alpha f - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Diagnostic Assay:

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and confirmed by Sanger sequencing performed by GENEWIZ, Inc.

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and those that were correctly assembled were confirmed using Sanger sequencing performed by GENEWIZ, Inc. Deletion of pobAR from the P. putida KT2440 genome using pCJ041 was confirmed by amplification of a 2094 bp product in diagnostic colony PCR using MyTaq™ HS Red Mix (Bioline) with primers oCJ298 (5′-ACCTTTCATCTGCGGACC-3′) and oCJ299 (5′-ATCTGTGGCACCCACTTG-3′) rather than the 3942 bp product generated when pobA and pobR are present.

    Clone Assay:

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: H19/IGF2 ICR amplicon was cloned using the pGEM T Easy Vector System ligation buffer protocol (Promega). .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and confirmed by Sanger sequencing performed by GENEWIZ, Inc.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The bacterial clones were screened using the PCR (PCR Master Mix, Thermo Fisher) following the manufacturer’s instructions.

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK). .. Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair.

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: .. All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA). .. Each vector was isolated and digested with a pair of respective restriction endonucleases to determine the presence of the insert.

    Article Title: Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing
    Article Snippet: The PCR amplicons were cloned using pGEM-T Easy Vector System (Promega) according to the manufacturer's instructions. .. Bacteria used were NEB 5-α F'Iq Competent E. coli (NEB).

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205). .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The bacterial clones were screened using the PCR (PCR Master Mix, Thermo Fisher) following the manufacturer’s instructions.

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and those that were correctly assembled were confirmed using Sanger sequencing performed by GENEWIZ, Inc. Deletion of pobAR from the P. putida KT2440 genome using pCJ041 was confirmed by amplification of a 2094 bp product in diagnostic colony PCR using MyTaq™ HS Red Mix (Bioline) with primers oCJ298 (5′-ACCTTTCATCTGCGGACC-3′) and oCJ299 (5′-ATCTGTGGCACCCACTTG-3′) rather than the 3942 bp product generated when pobA and pobR are present.

    Amplification:

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: H19/IGF2 ICR amplicon was cloned using the pGEM T Easy Vector System ligation buffer protocol (Promega). .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: To construct the suicide vector for this deletion, 1000 bp targeting regions 5’ and 3’ of the crc gene (PP_5292, GenBank: AE015451.1:6039987..6040766) were amplified from P. putida KT2440 gDNA using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primer pairs oCJ255 (5’-AACAGCTATGACATGATTACGAATTCGTAGCGTAGTGTGACTTGAAGGGCACAC-3’) & oCJ256 (5’-TTGCGGCCGCAAATGGCCCCATAAATCTCGTGCGTGTATG-3’) and oCJ257 (5’-GGGGCCATTTGCGGCCGCAAGGCCATTGGGGCTGCATTG-3’) & oCJ258 (5’-GCCTGCAGGTCGACTCTAGAGGATCCGCTTCCTCAAAGGCCAGGGC-3’), respectively, synthesized by Integrated DNA Technologies (IDT). .. These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Amplified products were then purified using the QIAquick PCR Purification kit (Qiagen) and digested with the corresponding enzyme (BsmBI, BbsI or SapI, New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: One µl of amplicon was cloned into Escherichia coli (DH5α) using the pGEM®-T Easy Vectors System (Promega, Madison, USA). .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK).

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: Similarly, the amplified Ligand-Hecate fragment was cloned into XhoI – NotI site of pKLAC2 ( ,B). .. All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA).

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205). .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Amplified products were then purified using the QIAquick PCR Purification kit (Qiagen) and digested with the corresponding enzyme (BsmBI, BbsI or SapI, New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: Similarly, pobA , encoding the 4-hydroxybenzoate hydroxylase, and a portion of pobR , which encodes the transcription factor that regulates expression of pobA , were deleted from the P. putida KT2440 genome to generate strain CJ182 using plasmid pCJ041. pCJ041 was constructed by amplifying a 1 kb 5′ targeting region with primer pair oCJ292 (5′-AGTGAGCGCAACGCAATTAATGTGAGTTAGCGAACTTTAGTAAAGGCTGGGCTTTCAGTTCATC-3′) and oCJ293 (5′-GCGGCCGCGGGCTGCGAGCTACGGG-3′) and a 1 kb 3′ targeting region with primer pair oCJ296 (5′-CCTGACCCGTAGCTCGCAGCCCGCGGCCGCGTGTGGATCAGCCGCCGTC-3’) and oCJ297 (5′-CCCTGAGTGCTTGCGGCAGCGTGAAGCTAGGCCCGCTTCGGTAAGGTCG-3’) from P. putida KT2440 gDNA and these were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions into pK18mobsacB (ATCC# 87097) ( , ) amplified linearly with primers oCJ288 (5′-CTAGCTTCACGCTGCCGCAAG-3′) and oCJ289 (5′-CTAACTCACATTAATTGCGTTGCGCTCACTG-3′). .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    DNA Synthesis:

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Plasmids were constructed using a combination of ligation of phosphorylated oligonucleotides, DNA synthesis by GenScript and IDT and Gibson Assembly.

    Synthesized:

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: To construct the suicide vector for this deletion, 1000 bp targeting regions 5’ and 3’ of the crc gene (PP_5292, GenBank: AE015451.1:6039987..6040766) were amplified from P. putida KT2440 gDNA using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primer pairs oCJ255 (5’-AACAGCTATGACATGATTACGAATTCGTAGCGTAGTGTGACTTGAAGGGCACAC-3’) & oCJ256 (5’-TTGCGGCCGCAAATGGCCCCATAAATCTCGTGCGTGTATG-3’) and oCJ257 (5’-GGGGCCATTTGCGGCCGCAAGGCCATTGGGGCTGCATTG-3’) & oCJ258 (5’-GCCTGCAGGTCGACTCTAGAGGATCCGCTTCCTCAAAGGCCAGGGC-3’), respectively, synthesized by Integrated DNA Technologies (IDT). .. These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Construct:

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: To construct the suicide vector for this deletion, 1000 bp targeting regions 5’ and 3’ of the crc gene (PP_5292, GenBank: AE015451.1:6039987..6040766) were amplified from P. putida KT2440 gDNA using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primer pairs oCJ255 (5’-AACAGCTATGACATGATTACGAATTCGTAGCGTAGTGTGACTTGAAGGGCACAC-3’) & oCJ256 (5’-TTGCGGCCGCAAATGGCCCCATAAATCTCGTGCGTGTATG-3’) and oCJ257 (5’-GGGGCCATTTGCGGCCGCAAGGCCATTGGGGCTGCATTG-3’) & oCJ258 (5’-GCCTGCAGGTCGACTCTAGAGGATCCGCTTCCTCAAAGGCCAGGGC-3’), respectively, synthesized by Integrated DNA Technologies (IDT). .. These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: .. All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA). .. Each vector was isolated and digested with a pair of respective restriction endonucleases to determine the presence of the insert.

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: Similarly, pobA , encoding the 4-hydroxybenzoate hydroxylase, and a portion of pobR , which encodes the transcription factor that regulates expression of pobA , were deleted from the P. putida KT2440 genome to generate strain CJ182 using plasmid pCJ041. pCJ041 was constructed by amplifying a 1 kb 5′ targeting region with primer pair oCJ292 (5′-AGTGAGCGCAACGCAATTAATGTGAGTTAGCGAACTTTAGTAAAGGCTGGGCTTTCAGTTCATC-3′) and oCJ293 (5′-GCGGCCGCGGGCTGCGAGCTACGGG-3′) and a 1 kb 3′ targeting region with primer pair oCJ296 (5′-CCTGACCCGTAGCTCGCAGCCCGCGGCCGCGTGTGGATCAGCCGCCGTC-3’) and oCJ297 (5′-CCCTGAGTGCTTGCGGCAGCGTGAAGCTAGGCCCGCTTCGGTAAGGTCG-3’) from P. putida KT2440 gDNA and these were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions into pK18mobsacB (ATCC# 87097) ( , ) amplified linearly with primers oCJ288 (5′-CTAGCTTCACGCTGCCGCAAG-3′) and oCJ289 (5′-CTAACTCACATTAATTGCGTTGCGCTCACTG-3′). .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were constructed using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs – NEB) or T4 DNA ligase (NEB) according to manufacturer's instructions. .. Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions.

    Incubation:

    Article Title: CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
    Article Snippet: .. T4 ligase was added, and the reaction containing digested gDNA and ligase was incubated overnight at 16°C, then transformed into 5-alpha F’I q High Efficiency Competent E . coli cells (NEB, C2992H), and transformants were selected on media supplemented with TMP. .. Lastly, “rescued” plasmids from multiple transformants were isolated and subjected to Sanger sequencing.

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions. .. The KvDMR1 amplicon was cloned using CopyControl PCR cloning kit with TransforMax™ EPI300™ Electrocompetent E. coli cells (Epicenter Biotechnologies) according to the manufacturer’s specifications except that all the incubation procedures were done at room temperature.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol. .. Transformants were recovered in 895 μL of SOC at 37 °C with shaking for 1 h. One hundered microliters of each recovery solution was spread onto LB agar plates and incubated at 37 °C overnight.

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Transformants were selected on LB (Miller) agar plates containing 50 mg/L kanamycin sulfate for selection and incubated at 37 °C.

    Expressing:

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: The E166G polymerase mutation was inserted into the expression plasmids using the Q5 site-directed mutagenesis kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA). .. All three pKLAC2 vectors (mPlum , Ligand-Hecate and plasmid-only ) were linearized with SacII to generate the expression cassettes.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: The E166G polymerase mutation was inserted into the expression plasmids using the Q5 site-directed mutagenesis kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: Similarly, pobA , encoding the 4-hydroxybenzoate hydroxylase, and a portion of pobR , which encodes the transcription factor that regulates expression of pobA , were deleted from the P. putida KT2440 genome to generate strain CJ182 using plasmid pCJ041. pCJ041 was constructed by amplifying a 1 kb 5′ targeting region with primer pair oCJ292 (5′-AGTGAGCGCAACGCAATTAATGTGAGTTAGCGAACTTTAGTAAAGGCTGGGCTTTCAGTTCATC-3′) and oCJ293 (5′-GCGGCCGCGGGCTGCGAGCTACGGG-3′) and a 1 kb 3′ targeting region with primer pair oCJ296 (5′-CCTGACCCGTAGCTCGCAGCCCGCGGCCGCGTGTGGATCAGCCGCCGTC-3’) and oCJ297 (5′-CCCTGAGTGCTTGCGGCAGCGTGAAGCTAGGCCCGCTTCGGTAAGGTCG-3’) from P. putida KT2440 gDNA and these were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions into pK18mobsacB (ATCC# 87097) ( , ) amplified linearly with primers oCJ288 (5′-CTAGCTTCACGCTGCCGCAAG-3′) and oCJ289 (5′-CTAACTCACATTAATTGCGTTGCGCTCACTG-3′). .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Transformation Assay:

    Article Title: CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
    Article Snippet: .. T4 ligase was added, and the reaction containing digested gDNA and ligase was incubated overnight at 16°C, then transformed into 5-alpha F’I q High Efficiency Competent E . coli cells (NEB, C2992H), and transformants were selected on media supplemented with TMP. .. Lastly, “rescued” plasmids from multiple transformants were isolated and subjected to Sanger sequencing.

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions. .. The KvDMR1 amplicon was cloned using CopyControl PCR cloning kit with TransforMax™ EPI300™ Electrocompetent E. coli cells (Epicenter Biotechnologies) according to the manufacturer’s specifications except that all the incubation procedures were done at room temperature.

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: .. These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. ..

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The bacterial clones were screened using the PCR (PCR Master Mix, Thermo Fisher) following the manufacturer’s instructions.

    Article Title: Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
    Article Snippet: .. NEB 5-alpha F’I q competent E.coli high efficiency cells (New England Biolabs, UK) were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours. .. Plasmids were extracted using Qiagen miniprep kit and mutations verified by sequencing (Eurofins Genomics).

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: .. Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol. .. Transformants were recovered in 895 μL of SOC at 37 °C with shaking for 1 h. One hundered microliters of each recovery solution was spread onto LB agar plates and incubated at 37 °C overnight.

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573). .. For generation of GST fusion proteins, full-length mouse Vdac1 (NM_011694.4), Tomm7 , Slc25a31 , Cox5b , Mdh2 , Pink1 ( ) and Pink1 epitopes were amplified by PCR and cloned in pGEX 4T1 (GE Healthcare Life Sciences, 28-9545-49).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The bacterial clones were screened using the PCR (PCR Master Mix, Thermo Fisher) following the manufacturer’s instructions.

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and those that were correctly assembled were confirmed using Sanger sequencing performed by GENEWIZ, Inc. Deletion of pobAR from the P. putida KT2440 genome using pCJ041 was confirmed by amplification of a 2094 bp product in diagnostic colony PCR using MyTaq™ HS Red Mix (Bioline) with primers oCJ298 (5′-ACCTTTCATCTGCGGACC-3′) and oCJ299 (5′-ATCTGTGGCACCCACTTG-3′) rather than the 3942 bp product generated when pobA and pobR are present.

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: .. Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Transformants were selected on LB (Miller) agar plates containing 50 mg/L kanamycin sulfate for selection and incubated at 37 °C.

    Electroporation:

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. The resulting plasmid, pCJ033, was transformed into KT2440-CJ102 by electroporation ( ).

    Transfection:

    Article Title: Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha
    Article Snippet: Plasmids pCK-Drosha-FLAG and pCK-V5-DGCR8 were obtained from Professor V. Narry Kim and propagated in Escherichia coli (strain JM109, [Stratagene] for pCK-V5-DGCR8 and 5 α F′ Iq C2992H [NEB] for pCK-Drosha-FLAG). .. Cells were harvested 48 h after transfection and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) containing three cOmplete Mini Protease Inhibitor Cocktail Tablets (Roche) per 25 mL lysis buffer.

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: Plasmids pRFP-nls or pMAX-GFP (Lonza, VPD-1001) were used as controls for transfection. .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Ligation:

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: H19/IGF2 ICR amplicon was cloned using the pGEM T Easy Vector System ligation buffer protocol (Promega). .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: .. Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol. .. Transformants were recovered in 895 μL of SOC at 37 °C with shaking for 1 h. One hundered microliters of each recovery solution was spread onto LB agar plates and incubated at 37 °C overnight.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Plasmids were constructed using a combination of ligation of phosphorylated oligonucleotides, DNA synthesis by GenScript and IDT and Gibson Assembly.

    Protease Inhibitor:

    Article Title: Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha
    Article Snippet: Plasmids pCK-Drosha-FLAG and pCK-V5-DGCR8 were obtained from Professor V. Narry Kim and propagated in Escherichia coli (strain JM109, [Stratagene] for pCK-V5-DGCR8 and 5 α F′ Iq C2992H [NEB] for pCK-Drosha-FLAG). .. Cells were harvested 48 h after transfection and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) containing three cOmplete Mini Protease Inhibitor Cocktail Tablets (Roche) per 25 mL lysis buffer.

    Introduce:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: RFP-nls was cloned into pCMV5-T7b (derivative of pCMV5 with T7 tag, D. Russell, University of Texas Southwestern) by using NotI and EcoRI to introduce the 3’ nuclear localization signals of RPS6/S6 ribosomal subunit, and EcoRI and NheI to place the RFP (pDS-Red N1; Clontech Laboratories, 632429) in frame 5’ of the nuclear localization signal, similarly previously GFP-nls was generated. .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Generated:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: RFP-nls was cloned into pCMV5-T7b (derivative of pCMV5 with T7 tag, D. Russell, University of Texas Southwestern) by using NotI and EcoRI to introduce the 3’ nuclear localization signals of RPS6/S6 ribosomal subunit, and EcoRI and NheI to place the RFP (pDS-Red N1; Clontech Laboratories, 632429) in frame 5’ of the nuclear localization signal, similarly previously GFP-nls was generated. .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and those that were correctly assembled were confirmed using Sanger sequencing performed by GENEWIZ, Inc. Deletion of pobAR from the P. putida KT2440 genome using pCJ041 was confirmed by amplification of a 2094 bp product in diagnostic colony PCR using MyTaq™ HS Red Mix (Bioline) with primers oCJ298 (5′-ACCTTTCATCTGCGGACC-3′) and oCJ299 (5′-ATCTGTGGCACCCACTTG-3′) rather than the 3942 bp product generated when pobA and pobR are present.

    Colony-forming Unit Assay:

    Article Title: Annexin A2 Modulates ROS and Impacts Inflammatory Response via IL-17 Signaling in Polymicrobial Sepsis Mice
    Article Snippet: .. E . coli (ATCC 25922) was bought from ATCC and NEB 5-alpha F'Iq Competent E . coli (C2992H) was bought from BioLabs Inc. Homogenates of peritoneal, blood, and other tissues were plated for colony forming units (CFU) assay [ , ]. .. Plasmid construction AnxA2 relevant genes were amplified from mice cDNA with specific primers by PCR and cloned into the PstI and XbaI sites of the PCAGGS-GFP vector (Addgene, Cambridge, MA) ( ).

    Polymerase Chain Reaction:

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: DNA Methylation analysis of the KvDMR1 and H19/IGF2 ICR Bisulfite-converted DNA amplicons were isolated from agarose gels using the Wizard SV gel and PCR Clean-Up System (Promega, Madison, WI). .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Amplified products were then purified using the QIAquick PCR Purification kit (Qiagen) and digested with the corresponding enzyme (BsmBI, BbsI or SapI, New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK). .. Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair.

    Article Title: Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
    Article Snippet: PCR was performed for 16–18 cycles using Pfu turbo polymerase and products digested with DpnI for 1 hour at 37 °C. .. NEB 5-alpha F’I q competent E.coli high efficiency cells (New England Biolabs, UK) were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: The digested PCR products (triazole and normal) and linearized plasmid were ligated for 16 h at 15 °C (total volume 10 μL, 1∶3 vector∶insert ratio) using T4 DNA ligase (Promega, Cat. No. M1801). .. Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol.

    Article Title: Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing
    Article Snippet: The PCR amplicons were cloned using pGEM-T Easy Vector System (Promega) according to the manufacturer's instructions. .. Bacteria used were NEB 5-α F'Iq Competent E. coli (NEB).

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205). .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Amplified products were then purified using the QIAquick PCR Purification kit (Qiagen) and digested with the corresponding enzyme (BsmBI, BbsI or SapI, New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: These PCR amplifications were performed using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs). .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Recombinant:

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: Paragraph title: Genetic engineering of K. lactis to express recombinant proteins ... All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA).

    DNA Extraction:

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: Paragraph title: DNA Extraction and Sequencing of the ITS1-5.8S-ITS2 Region ... The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK).

    Mutagenesis:

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: The E166G polymerase mutation was inserted into the expression plasmids using the Q5 site-directed mutagenesis kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
    Article Snippet: Paragraph title: Mutagenesis ... NEB 5-alpha F’I q competent E.coli high efficiency cells (New England Biolabs, UK) were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours.

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: Paragraph title: Plasmids, primers, mutagenesis ... Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: The E166G polymerase mutation was inserted into the expression plasmids using the Q5 site-directed mutagenesis kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Isolation:

    Article Title: CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
    Article Snippet: T4 ligase was added, and the reaction containing digested gDNA and ligase was incubated overnight at 16°C, then transformed into 5-alpha F’I q High Efficiency Competent E . coli cells (NEB, C2992H), and transformants were selected on media supplemented with TMP. .. Lastly, “rescued” plasmids from multiple transformants were isolated and subjected to Sanger sequencing.

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: DNA Methylation analysis of the KvDMR1 and H19/IGF2 ICR Bisulfite-converted DNA amplicons were isolated from agarose gels using the Wizard SV gel and PCR Clean-Up System (Promega, Madison, WI). .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA). .. Each vector was isolated and digested with a pair of respective restriction endonucleases to determine the presence of the insert.

    Purification:

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK). .. Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Sequencing:

    Article Title: CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
    Article Snippet: T4 ligase was added, and the reaction containing digested gDNA and ligase was incubated overnight at 16°C, then transformed into 5-alpha F’I q High Efficiency Competent E . coli cells (NEB, C2992H), and transformants were selected on media supplemented with TMP. .. Lastly, “rescued” plasmids from multiple transformants were isolated and subjected to Sanger sequencing.

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and confirmed by Sanger sequencing performed by GENEWIZ, Inc.

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: Paragraph title: DNA Extraction and Sequencing of the ITS1-5.8S-ITS2 Region ... The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK).

    Article Title: Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
    Article Snippet: NEB 5-alpha F’I q competent E.coli high efficiency cells (New England Biolabs, UK) were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours. .. Plasmids were extracted using Qiagen miniprep kit and mutations verified by sequencing (Eurofins Genomics).

    Article Title: Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing
    Article Snippet: Both amplicons spanned SNPs identified by the whole genome sequencing of the B. t. indicus sire. .. Bacteria used were NEB 5-α F'Iq Competent E. coli (NEB).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions. .. Clones were screened by diagnostic restriction digest and those that were correctly assembled were confirmed using Sanger sequencing performed by GENEWIZ, Inc. Deletion of pobAR from the P. putida KT2440 genome using pCJ041 was confirmed by amplification of a 2094 bp product in diagnostic colony PCR using MyTaq™ HS Red Mix (Bioline) with primers oCJ298 (5′-ACCTTTCATCTGCGGACC-3′) and oCJ299 (5′-ATCTGTGGCACCCACTTG-3′) rather than the 3942 bp product generated when pobA and pobR are present.

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Sequences of all plasmids were confirmed using Sanger sequencing performed by GenScript or Eurofins Genomics.

    Protein Extraction:

    Article Title: Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha
    Article Snippet: Plasmids pCK-Drosha-FLAG and pCK-V5-DGCR8 were obtained from Professor V. Narry Kim and propagated in Escherichia coli (strain JM109, [Stratagene] for pCK-V5-DGCR8 and 5 α F′ Iq C2992H [NEB] for pCK-Drosha-FLAG). .. Cells were harvested 48 h after transfection and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) containing three cOmplete Mini Protease Inhibitor Cocktail Tablets (Roche) per 25 mL lysis buffer.

    Gel Extraction:

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK). .. Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Restriction products were gel purified using agarose (Thermo Fisher) and the QIAquick Gel Extraction Kit (Qiagen) then ligated using the Quick Ligation Kit (New England Biolabs). .. Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs).

    Plasmid Preparation:

    Article Title: CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon
    Article Snippet: Paragraph title: Plasmid rescue ... T4 ligase was added, and the reaction containing digested gDNA and ligase was incubated overnight at 16°C, then transformed into 5-alpha F’I q High Efficiency Competent E . coli cells (NEB, C2992H), and transformants were selected on media supplemented with TMP.

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: .. The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions. .. The KvDMR1 amplicon was cloned using CopyControl PCR cloning kit with TransforMax™ EPI300™ Electrocompetent E. coli cells (Epicenter Biotechnologies) according to the manufacturer’s specifications except that all the incubation procedures were done at room temperature.

    Article Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440
    Article Snippet: To construct the suicide vector for this deletion, 1000 bp targeting regions 5’ and 3’ of the crc gene (PP_5292, GenBank: AE015451.1:6039987..6040766) were amplified from P. putida KT2440 gDNA using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primer pairs oCJ255 (5’-AACAGCTATGACATGATTACGAATTCGTAGCGTAGTGTGACTTGAAGGGCACAC-3’) & oCJ256 (5’-TTGCGGCCGCAAATGGCCCCATAAATCTCGTGCGTGTATG-3’) and oCJ257 (5’-GGGGCCATTTGCGGCCGCAAGGCCATTGGGGCTGCATTG-3’) & oCJ258 (5’-GCCTGCAGGTCGACTCTAGAGGATCCGCTTCCTCAAAGGCCAGGGC-3’), respectively, synthesized by Integrated DNA Technologies (IDT). .. These fragments were then assembled into pK18mobsacB (ATCC 87097) ( ) digested with EcoRI and BamHI using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The positive clones were grown in 50 ml LB broth (Thermo Fisher), then the plasmids were extracted using the Plasmid Plus Midi Kit (Qiagen).

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK). .. Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair.

    Article Title: Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
    Article Snippet: Mutagenesis Point mutations were introduced into the WT hP2X7 plasmid using the Stratagene Quikchange II site-directed mutagenesis kit (Agilent Technologies). .. NEB 5-alpha F’I q competent E.coli high efficiency cells (New England Biolabs, UK) were transformed with 5 µl of digested product and colonies selected following growth at 37 °C for 16 hours.

    Article Title: Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts
    Article Snippet: For control, pKLAC2 without any foreign gene (plasmid-only ) was included. .. All three constructs were cloned into competent E. coli cells (NEB # C2992, New England Biolabs Inc., MA, USA).

    Article Title: Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha
    Article Snippet: Plasmids pCK-Drosha-FLAG and pCK-V5-DGCR8 were obtained from Professor V. Narry Kim and propagated in Escherichia coli (strain JM109, [Stratagene] for pCK-V5-DGCR8 and 5 α F′ Iq C2992H [NEB] for pCK-Drosha-FLAG). .. To generate a cell lysate for microprocessor-free controls, HEK293T cells were treated with PEI in the absence of plasmid.

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: The digested PCR products (triazole and normal) and linearized plasmid were ligated for 16 h at 15 °C (total volume 10 μL, 1∶3 vector∶insert ratio) using T4 DNA ligase (Promega, Cat. No. M1801). .. Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol.

    Article Title: Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing
    Article Snippet: The PCR amplicons were cloned using pGEM-T Easy Vector System (Promega) according to the manufacturer's instructions. .. Bacteria used were NEB 5-α F'Iq Competent E. coli (NEB).

    Article Title: Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs
    Article Snippet: Ligated products were transformed into the NEB 10-β or NEB 5-α F’Iq Competent E. coli (New England Biolabs). .. The positive clones were grown in 50 ml LB broth (Thermo Fisher), then the plasmids were extracted using the Plasmid Plus Midi Kit (Qiagen).

    Article Title: A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
    Article Snippet: Similarly, pobA , encoding the 4-hydroxybenzoate hydroxylase, and a portion of pobR , which encodes the transcription factor that regulates expression of pobA , were deleted from the P. putida KT2440 genome to generate strain CJ182 using plasmid pCJ041. pCJ041 was constructed by amplifying a 1 kb 5′ targeting region with primer pair oCJ292 (5′-AGTGAGCGCAACGCAATTAATGTGAGTTAGCGAACTTTAGTAAAGGCTGGGCTTTCAGTTCATC-3′) and oCJ293 (5′-GCGGCCGCGGGCTGCGAGCTACGGG-3′) and a 1 kb 3′ targeting region with primer pair oCJ296 (5′-CCTGACCCGTAGCTCGCAGCCCGCGGCCGCGTGTGGATCAGCCGCCGTC-3’) and oCJ297 (5′-CCCTGAGTGCTTGCGGCAGCGTGAAGCTAGGCCCGCTTCGGTAAGGTCG-3’) from P. putida KT2440 gDNA and these were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions into pK18mobsacB (ATCC# 87097) ( , ) amplified linearly with primers oCJ288 (5′-CTAGCTTCACGCTGCCGCAAG-3′) and oCJ289 (5′-CTAACTCACATTAATTGCGTTGCGCTCACTG-3′). .. The assemblies were transformed into NEB® 5-alpha F'I q competent Escherichia coli (New England Biolabs) according to the manufacturer's instructions.

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Paragraph title: Plasmid construction ... Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions.

    Software:

    Article Title: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli
    Article Snippet: Five microliters of each ligation mixture was transformed into chemically competent E. coli (NEB 5α, NEB, Cat. No. C2992H) using the standard protocol. .. Colonies were counted using a Gel Doc XR+ system and Quantity One Software (both from BioRad Laboratories).

    Negative Control:

    Article Title: Phylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates
    Article Snippet: A negative control was included in each set of PCR reactions. .. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F’Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM 4-TOPO® vector system (Invitrogen, Paisley, UK).

    Selection:

    Article Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
    Article Snippet: Plasmids were transformed into either competent Top10 (Life Technologies), NEB 5-alpha F’Iq (NEB), or Epi400 (Epicentre Biotechnologies) Escherichia coli according to manufacturer's instructions. .. Transformants were selected on LB (Miller) agar plates containing 50 mg/L kanamycin sulfate for selection and incubated at 37 °C.

    DNA Methylation Assay:

    Article Title: Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine
    Article Snippet: Paragraph title: DNA Methylation analysis of the KvDMR1 and H19/IGF2 ICR ... The plasmid was transformed into chemically competent NEB 5-alpha F’Iq E.Coli cells (New England BioLabs; Ipswich, MA) according to the manufacturer’s instructions.

    Article Title: Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing
    Article Snippet: Paragraph title: DNA methylation analysis at GNAS/GNASXL domain ... Bacteria used were NEB 5-α F'Iq Competent E. coli (NEB).

    Alkaline Lysis:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: .. Plasmids were transformed in E. coli bacteria NEB 5-alpha (New England Biolabs, C2992) or Xl10-GOLD (Agilent Technologies, 200314) and extracted by alkaline lysis with Nucleobond PC100 Midiprep kits (Macherey-Nagel, 740573). .. For generation of GST fusion proteins, full-length mouse Vdac1 (NM_011694.4), Tomm7 , Slc25a31 , Cox5b , Mdh2 , Pink1 ( ) and Pink1 epitopes were amplified by PCR and cloned in pGEX 4T1 (GE Healthcare Life Sciences, 28-9545-49).

    Lysis:

    Article Title: Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha
    Article Snippet: Plasmids pCK-Drosha-FLAG and pCK-V5-DGCR8 were obtained from Professor V. Narry Kim and propagated in Escherichia coli (strain JM109, [Stratagene] for pCK-V5-DGCR8 and 5 α F′ Iq C2992H [NEB] for pCK-Drosha-FLAG). .. Cells were harvested 48 h after transfection and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) containing three cOmplete Mini Protease Inhibitor Cocktail Tablets (Roche) per 25 mL lysis buffer.

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    New England Biolabs neb 5 alpha f
    Neb 5 Alpha F, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neb 5 alpha f/product/New England Biolabs
    Average 95 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    neb 5 alpha f - by Bioz Stars, 2020-02
    95/100 stars
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