antartic phosphatase  (New England Biolabs)


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    Structured Review

    New England Biolabs antartic phosphatase
    Antartic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antartic phosphatase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antartic phosphatase - by Bioz Stars, 2020-02
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product was digested by Bam HI and Kpn I, cloned into pGTP_FZ301 previously digested by Bam HI and Kpn I, and ligation was used to transform MG1363. pGTP_FZ301_NucB plasmid extracted from a chloramphenicol resistant clone was verified by digestion and sequencing. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: Generation of biotin linked PTENpg1 asRNAs PTENpg1 asRNA α was PCR amplified using primers with Nhe1 or Kpn1 restriction sites ( ) and cloned into pcDNA 3.1. .. The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. The PCR fragment was cloned as a BglII-BamH1 into pKgBΔSS and the resulting plasmid was labeled pKgBPR.

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: Paragraph title: Cloning of K. pneumoniae dedA ... For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: .. Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Constructs were transformed into competent bacteria and grown onto ampicilline agar plate to generate bacteria clones containing either the WT or the del18 construct.

    Amplification:

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: Generation of biotin linked PTENpg1 asRNAs PTENpg1 asRNA α was PCR amplified using primers with Nhe1 or Kpn1 restriction sites ( ) and cloned into pcDNA 3.1. .. The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs).

    Article Title: Early-onset primary antibody deficiency resembling common variable immunodeficiency challenges the diagnosis of Wiedeman-Steiner and Roifman syndromes
    Article Snippet: Genomic DNA was amplified by PCR using the specific primers and KAPA2G Robust Hotstart Ready Mix (KAPA Biosystems). .. PCR products were enzymatically purified with Exonuclease I and Antartic phosphatase (both New England BioLabs Inc.).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: .. DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. This plasmid was designated pKgBΔSS.

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: .. For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette. .. In parallel, the AprR cassette of pIJ773 was amplified using primers Apr1 and Apr2, digested with XbaI and ligated separately with the linearized plasmids described above using T4 ligase (Thermo Scientific, USA).

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Splicing Analysis PCR products containing either the Wild type (WT) or the c.393_410del18 (del18) mutation were amplified using primers containining both of them an artificial site for Kpn1 (sequences can be asked on requests) and as template a DNA sample of the patient heterozygous for the mutation. .. Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.).

    De-Phosphorylation Assay:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: .. To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. A pair of scarlet- targeting oligonucleotides were then annealed and ligated into the linearized pDR274 vector using a ligation mix (TaKaRa Bio, Shiga, Japan).

    Synthesized:

    Article Title: Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase
    Article Snippet: Dpn I, Eco RI HF and Not I HF restriction enzymes, T4 DNA ligase and Antartic Phosphatase were purchased from New England Biolabs (Beverly, MA, U.S.A.). .. Oligonucleotides were synthesized by Eurogenetec (Liege, Belgium).

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences). .. Dephosphorylated ribozymes (10 pmol), or chemically synthesized RNA substrates, were 5′-end-radiolabeled using 3 U of T4 polynucleotide kinase (Promega) for 1 h at 37°C in the presence of 3.2 pmol of [γ-32 P]ATP (6000 Ci/mmol; New England Nuclear).

    Autoradiography:

    Article Title: 5?-UTR G-quadruplex structures acting as translational repressors
    Article Snippet: RNA labeling In order to produce 5′-end-labeled PG4s, purified transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BioLabs,) to 50 pmol of RNA and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 µl containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20 U, Invitrogen). .. The bands of the correct sizes containing the 5′-end-labeled RNAs were excised and recovered as described above except that the detection was performed by autoradiography.

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences). .. The reactions were stopped by the addition of ice-cold formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol), and the RNA molecules were purified by 8%–20% polyacrylamide gel electrophoresis and recovered as described above except that the detection was performed by autoradiography.

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: sgRNA labeling and in-line probing To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. 5′-end-labeled sgRNAs were visualized by autoradiography and the bands corresponding to the correct sizes were excised from the gel, gel slices shredded and the transcripts eluted for 10 min at 37°C in 300 μL of water.

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV
    Article Snippet: RNA Substrate Labeling In order to generate 5′-end-labeled RNA substrate, 50 pmol of purified transcripts were dephosphorylated by adding 1 U of Antartic phosphatase (New England Biolabs) and incubating for 1 h at 37°C in a final volume of 10 µL containing 50 mM Bis-Propane, pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and 40 U of RNAGuard. .. The 5′-end-labeled RNA substrates were fractionated on 5% PAGE gels and the RNAs detected by autoradiography, cut out and eluted as described above.

    Construct:

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: Cloning of K. pneumoniae dedA The K. pneumoniae dedA gene (KpndedA ) was amplified from RH201207 genomic DNA using primers KdedA1 and KdedA2 , digested with SacI and HindIII (New England Biolabs, UAS) and ligated with a similarly digested and dephosphorylated linearized fragment of vector pBAD-HisA (Invitrogen, USA) to construct plasmid pBAD-KpndedA . .. For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Constructs were transformed into competent bacteria and grown onto ampicilline agar plate to generate bacteria clones containing either the WT or the del18 construct.

    Electrophoresis:

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences). .. Radiolabelled transcripts were purified by electrophoresis on a denaturing gel and recovered as described above.

    Incubation:

    Article Title: From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors
    Article Snippet: Binding of Radioactive Aptamers on Adherent Cells First, 2′F-Py RNAs were dephosphorylated at the 5′-end using antartic phosphatase (New England Biolabs) before 5′-[32 P] labeling (3×103 Bq.pmole−1 ) using T4 kinase. .. Radioactive aptamers at different concentrations were incubated for 15 min at 37°C on 80% confluent cell monolayer in 200 µL of RPMI 1640 containing 100 µg/ml of tRNA as a nonspecific competitor.

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences). .. For the 3′-end labeling of ribozymes, 15 pmol were incubated for 1 h at 37°C in a final volume of 10 μL containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 10% dimethylsulfoxide, 10 pmol [32 P]Cp (3000 Ci/mmol; New England Nuclear), and 10 U of T4 RNA ligase (New England Biolabs).

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: Each NTP (5 mM) was added to the mixture either with or without 50 µCi of [α-32 P]UTP (3 000 Ci/mmol, New England Nuclear) in a final volume of 100 µL, and the reaction incubated at 37°C for 2 to 4 h. Upon completion, the reaction mixtures were treated with DNase RQ1 (Promega) at 37°C for 20 min, and the RNA then purified by phenol:chloroform extraction and precipitation with ethanol. .. In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences).

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV
    Article Snippet: RNA Substrate Labeling In order to generate 5′-end-labeled RNA substrate, 50 pmol of purified transcripts were dephosphorylated by adding 1 U of Antartic phosphatase (New England Biolabs) and incubating for 1 h at 37°C in a final volume of 10 µL containing 50 mM Bis-Propane, pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and 40 U of RNAGuard. .. The dephosphorylated RNAs (5 pmol) were then 5′-end labeled by incubation for 1 h at 37°C with 3 U of T4 polynucleotide kinase (USB) and 3.2 pmol of [γ-32P ]ATP (6000 Ci/mmol; New England Nuclear) in the provided reaction buffer.

    Expressing:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PZn zitR SPExp4 expression and secretion cassette was PCR-amplified from pGTPb_PZn zitR- SPExp4 using primers 7 and 8. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: .. To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. A pair of scarlet- targeting oligonucleotides were then annealed and ligated into the linearized pDR274 vector using a ligation mix (TaKaRa Bio, Shiga, Japan).

    Article Title: Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase
    Article Snippet: E.coli TOP 10 electrocompetent cells (Invitrogen, Carlsbad, U.S.A.) were utilized as gene expression and production of recombinant amylosucrase variants. .. Dpn I, Eco RI HF and Not I HF restriction enzymes, T4 DNA ligase and Antartic Phosphatase were purchased from New England Biolabs (Beverly, MA, U.S.A.).

    Transformation Assay:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: Ligation reaction was transformed into NEB 5-α competent cells. pGTPb_PZn zitR- SPExp4 from an ampicillin-resistant clone was verified by digestion and sequencing, revealing three silent mutations in zitR sequence. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Constructs were transformed into competent bacteria and grown onto ampicilline agar plate to generate bacteria clones containing either the WT or the del18 construct.

    Derivative Assay:

    Article Title: Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase
    Article Snippet: Plasmid pGST‐amylosucrase (pGST‐AS) derived from the pGEX‐6P‐3 (GE Healthcare Biosciences, Piscataway, U.S.A) encoding GST fused to N. polysaccharea amylosucrase, was used for the construction of semi‐rational libraries. .. Dpn I, Eco RI HF and Not I HF restriction enzymes, T4 DNA ligase and Antartic Phosphatase were purchased from New England Biolabs (Beverly, MA, U.S.A.).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: Plasmid pKΔ4B was derived by Cai et al. following engineering of linker-insertion mutants in the glycoprotein B gene. .. DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ).

    Flow Cytometry:

    Article Title: Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC-UV-MS
    Article Snippet: Nucleobond AXR-80 gravity flow columns were purchased from Machery-Nagel (Bethlehem, PA). .. Snake venom phosphodiesterase I was purchased from Worthington Biochemicals (Lakewood, NJ) and antartic phosphatase from New England Biolabs (Ipswich, MA).

    Ligation:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product was digested by Bam HI and Kpn I, cloned into pGTP_FZ301 previously digested by Bam HI and Kpn I, and ligation was used to transform MG1363. pGTP_FZ301_NucB plasmid extracted from a chloramphenicol resistant clone was verified by digestion and sequencing. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. A pair of scarlet- targeting oligonucleotides were then annealed and ligated into the linearized pDR274 vector using a ligation mix (TaKaRa Bio, Shiga, Japan).

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: .. Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Constructs were transformed into competent bacteria and grown onto ampicilline agar plate to generate bacteria clones containing either the WT or the del18 construct.

    Generated:

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: T7 transcripts were generated by linearization of pcDNA 3.1 with Kpn1 and T7 transcribed with the Durascribe T7 kit (Epicenter, Madison WI, USA) using fluorinated CTPs and UTPs (TriLink). .. The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs).

    Sequencing:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product was digested by Bam HI and Kpn I, cloned into pGTP_FZ301 previously digested by Bam HI and Kpn I, and ligation was used to transform MG1363. pGTP_FZ301_NucB plasmid extracted from a chloramphenicol resistant clone was verified by digestion and sequencing. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: Early-onset primary antibody deficiency resembling common variable immunodeficiency challenges the diagnosis of Wiedeman-Steiner and Roifman syndromes
    Article Snippet: Paragraph title: Sanger sequencing of genomic DNA ... PCR products were enzymatically purified with Exonuclease I and Antartic phosphatase (both New England BioLabs Inc.).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. The sequence of gB amino acids spanning 711 to 796 were deleted from pKgBΔSS by cassette PCR mutagenesis.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Midiprep Purified (Macherey-Nagel) constructs were then verified by sequencing.

    Antiviral Assay:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product digested by Ava II was cloned into pGTPb_102a_SPExp4 previously digested by Rsr II and dephosphorylated. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Recombinant:

    Article Title: Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase
    Article Snippet: E.coli TOP 10 electrocompetent cells (Invitrogen, Carlsbad, U.S.A.) were utilized as gene expression and production of recombinant amylosucrase variants. .. Dpn I, Eco RI HF and Not I HF restriction enzymes, T4 DNA ligase and Antartic Phosphatase were purchased from New England Biolabs (Beverly, MA, U.S.A.).

    In Vivo:

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs). .. For in vivo pulldown, MCF7 cells were transfected, 30h later collected and avidin IPs performed as described , .

    Radioactivity:

    Article Title: From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors
    Article Snippet: Binding of Radioactive Aptamers on Adherent Cells First, 2′F-Py RNAs were dephosphorylated at the 5′-end using antartic phosphatase (New England Biolabs) before 5′-[32 P] labeling (3×103 Bq.pmole−1 ) using T4 kinase. .. After five washings with 500 µL of culture medium, bound sequences were recovered in 200 µL of lysis buffer (50 mM Hepes PH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X100, 2 mM MgCl2 and 5 mM EGTA) and the radioactivity counted.

    Mutagenesis:

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. The sequence of gB amino acids spanning 711 to 796 were deleted from pKgBΔSS by cassette PCR mutagenesis.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Splicing Analysis PCR products containing either the Wild type (WT) or the c.393_410del18 (del18) mutation were amplified using primers containining both of them an artificial site for Kpn1 (sequences can be asked on requests) and as template a DNA sample of the patient heterozygous for the mutation. .. Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.).

    Transfection:

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs). .. The T7 transcripts were transfected into target cells at 25-50nM using Lipofectamine 2000 (Life Technologies).

    Labeling:

    Article Title: From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors
    Article Snippet: .. Binding of Radioactive Aptamers on Adherent Cells First, 2′F-Py RNAs were dephosphorylated at the 5′-end using antartic phosphatase (New England Biolabs) before 5′-[32 P] labeling (3×103 Bq.pmole−1 ) using T4 kinase. ..

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. The PCR fragment was cloned as a BglII-BamH1 into pKgBΔSS and the resulting plasmid was labeled pKgBPR.

    Article Title: 5?-UTR G-quadruplex structures acting as translational repressors
    Article Snippet: .. RNA labeling In order to produce 5′-end-labeled PG4s, purified transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BioLabs,) to 50 pmol of RNA and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 µl containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20 U, Invitrogen). .. Dephosphorylated transcripts (5 pmol) were 5′-end-radiolabeled using 3 U of T4 polynucleotide kinase (Promega) for 1 h at 37°C in the presence of 3.2 pmol of [α-32 P]ATP (6000 Ci/mmol; New England Nuclear).

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: Paragraph title: RNA labeling ... In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences).

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: .. sgRNA labeling and in-line probing To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. Dephosphorylated sgRNAs (5 pmol) were 5′-end-rediolabeled using 3 U of T4 polynucleotide kinase (New England Biolabs) for 1 h at 37°C in the presence of 3.2 pmol of [α-32 P]ATP (6000 Ci/mmol; Perkin Elmer).

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV
    Article Snippet: .. RNA Substrate Labeling In order to generate 5′-end-labeled RNA substrate, 50 pmol of purified transcripts were dephosphorylated by adding 1 U of Antartic phosphatase (New England Biolabs) and incubating for 1 h at 37°C in a final volume of 10 µL containing 50 mM Bis-Propane, pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and 40 U of RNAGuard. .. The dephosphorylated RNAs (5 pmol) were then 5′-end labeled by incubation for 1 h at 37°C with 3 U of T4 polynucleotide kinase (USB) and 3.2 pmol of [γ-32P ]ATP (6000 Ci/mmol; New England Nuclear) in the provided reaction buffer.

    Avidin-Biotin Assay:

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs). .. For in vivo pulldown, MCF7 cells were transfected, 30h later collected and avidin IPs performed as described , .

    Polymerase Chain Reaction:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product was digested by Bam HI and Kpn I, cloned into pGTP_FZ301 previously digested by Bam HI and Kpn I, and ligation was used to transform MG1363. pGTP_FZ301_NucB plasmid extracted from a chloramphenicol resistant clone was verified by digestion and sequencing. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells
    Article Snippet: Generation of biotin linked PTENpg1 asRNAs PTENpg1 asRNA α was PCR amplified using primers with Nhe1 or Kpn1 restriction sites ( ) and cloned into pcDNA 3.1. .. The resultant transcripts were DNase treated and dephosporylation using Antartic Phosphatase (New England Biolabs).

    Article Title: Early-onset primary antibody deficiency resembling common variable immunodeficiency challenges the diagnosis of Wiedeman-Steiner and Roifman syndromes
    Article Snippet: .. PCR products were enzymatically purified with Exonuclease I and Antartic phosphatase (both New England BioLabs Inc.). .. Purified PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) on a 3730xl DNA Analyzer (Applied Biosystems).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: .. DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ). .. This plasmid was designated pKgBΔSS.

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: PCR amplicons of 401 bp containing the entire exon 2 and exon-intron boundaries of Phox2b (188 bp) starting 112 bp upstream and finishing 101 bp downstream of Phox2b exon2 respectively. .. Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.).

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: Briefly, transcriptions were performed using purified T7 RNA polymerase (10 µg) in the presence of RNA Guard (24 U, Amersham Biosciences) and pyrophosphatase (0.01 U, Roche Diagnostics) in a buffer containing 80 mM HEPES-KOH, pH 7.5, 24 mM MgCl2 , 2 mM spermidine, 40 mM DTT and either linearized plasmid DNA (5 µg) or PCR product (100 pmol) as template. .. In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences).

    Binding Assay:

    Article Title: From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors
    Article Snippet: .. Binding of Radioactive Aptamers on Adherent Cells First, 2′F-Py RNAs were dephosphorylated at the 5′-end using antartic phosphatase (New England Biolabs) before 5′-[32 P] labeling (3×103 Bq.pmole−1 ) using T4 kinase. ..

    Polyacrylamide Gel Electrophoresis:

    Article Title: 5?-UTR G-quadruplex structures acting as translational repressors
    Article Snippet: RNA labeling In order to produce 5′-end-labeled PG4s, purified transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BioLabs,) to 50 pmol of RNA and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 µl containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20 U, Invitrogen). .. The reactions were stopped by adding formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol), and the RNA molecules purified by 10% polyacrylamide gel electrophoresis.

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences). .. The reactions were stopped by the addition of ice-cold formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol), and the RNA molecules were purified by 8%–20% polyacrylamide gel electrophoresis and recovered as described above except that the detection was performed by autoradiography.

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: sgRNA labeling and in-line probing To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. The reaction was stopped by adding formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol), and the radiolabeled sgRNA purified by 8% polyacrylamide gel electrophoresis.

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: RNA products were fractionated by a denaturing (7 M urea) 6 to 20% polyacrylamide gel electrophoresis (PAGE; 19∶1 ratio of acrylamide to bisacrylamide) using 45 mM Tris-borate, pH 7.5/1 mM EDTA solution as running buffer. .. In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences).

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV
    Article Snippet: RNA Substrate Labeling In order to generate 5′-end-labeled RNA substrate, 50 pmol of purified transcripts were dephosphorylated by adding 1 U of Antartic phosphatase (New England Biolabs) and incubating for 1 h at 37°C in a final volume of 10 µL containing 50 mM Bis-Propane, pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and 40 U of RNAGuard. .. The 5′-end-labeled RNA substrates were fractionated on 5% PAGE gels and the RNAs detected by autoradiography, cut out and eluted as described above.

    CRISPR:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: Paragraph title: Knockout of Scarlet by CRISPR/Cas9 system ... To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA).

    IA:

    Article Title: Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC-UV-MS
    Article Snippet: Snake venom phosphodiesterase I was purchased from Worthington Biochemicals (Lakewood, NJ) and antartic phosphatase from New England Biolabs (Ipswich, MA). .. Nanopure water (18 Mohms) from a Barnstead (Dubuque, IA) filtering system was autoclaved and used in all buffers and solutions.

    Purification:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. To synthesize gRNAs in vitro , gRNA synthesis vectors were digested by Dra I (NEW ENGLAND Biolabs, Connecticut, USA) and purified by phenol/chloroform extraction.

    Article Title: Early-onset primary antibody deficiency resembling common variable immunodeficiency challenges the diagnosis of Wiedeman-Steiner and Roifman syndromes
    Article Snippet: .. PCR products were enzymatically purified with Exonuclease I and Antartic phosphatase (both New England BioLabs Inc.). .. Purified PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) on a 3730xl DNA Analyzer (Applied Biosystems).

    Article Title: Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease
    Article Snippet: Amplicons were then digested with Asp718 (iso -schizomer of KpnI) for convenient reasons to subclone it into the Exontrap cloning system (MoBiTec) at the multiple cloning site previously digested with Asp718 and dephosphorylated using antartic phosphatase (New England Biolabs inc.) before ligation with the T4 DNA ligase (New England Biolab inc.). .. Midiprep Purified (Macherey-Nagel) constructs were then verified by sequencing.

    Article Title: 5?-UTR G-quadruplex structures acting as translational repressors
    Article Snippet: .. RNA labeling In order to produce 5′-end-labeled PG4s, purified transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BioLabs,) to 50 pmol of RNA and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 µl containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20 U, Invitrogen). .. Dephosphorylated transcripts (5 pmol) were 5′-end-radiolabeled using 3 U of T4 polynucleotide kinase (Promega) for 1 h at 37°C in the presence of 3.2 pmol of [α-32 P]ATP (6000 Ci/mmol; New England Nuclear).

    Article Title: Potassium ions modulate a G-quadruplex-ribozyme's activity
    Article Snippet: In order to produce 5′-end-labeled ribozymes, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 50 pmol of ribozyme and incubating the reaction mixture for 30 min at 37°C in a final volume of 10 μL containing 50 mM Bis-Propane (pH 6.0), 1 mM MgCl2 , 0.1 mM ZnCl2 , and 40 U of RNAGuard (Amersham Biosciences). .. The reactions were stopped by the addition of ice-cold formamide dye buffer (95% formamide, 10 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol), and the RNA molecules were purified by 8%–20% polyacrylamide gel electrophoresis and recovered as described above except that the detection was performed by autoradiography.

    Article Title: CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo
    Article Snippet: .. sgRNA labeling and in-line probing To produce 5′-end-labeled sgRNAs, purified sgRNA transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England BIoLabs) to 50 pmol of sgRNA in a final volume of 10 μL containing 50 mM Bis-Propane pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and RNase OUT (20U, Invitrogen). .. Dephosphorylated sgRNAs (5 pmol) were 5′-end-rediolabeled using 3 U of T4 polynucleotide kinase (New England Biolabs) for 1 h at 37°C in the presence of 3.2 pmol of [α-32 P]ATP (6000 Ci/mmol; Perkin Elmer).

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: Each NTP (5 mM) was added to the mixture either with or without 50 µCi of [α-32 P]UTP (3 000 Ci/mmol, New England Nuclear) in a final volume of 100 µL, and the reaction incubated at 37°C for 2 to 4 h. Upon completion, the reaction mixtures were treated with DNase RQ1 (Promega) at 37°C for 20 min, and the RNA then purified by phenol:chloroform extraction and precipitation with ethanol. .. In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences).

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV
    Article Snippet: .. RNA Substrate Labeling In order to generate 5′-end-labeled RNA substrate, 50 pmol of purified transcripts were dephosphorylated by adding 1 U of Antartic phosphatase (New England Biolabs) and incubating for 1 h at 37°C in a final volume of 10 µL containing 50 mM Bis-Propane, pH 6.0, 1 mM MgCl2 , 0.1 mM ZnCl2 and 40 U of RNAGuard. .. The dephosphorylated RNAs (5 pmol) were then 5′-end labeled by incubation for 1 h at 37°C with 3 U of T4 polynucleotide kinase (USB) and 3.2 pmol of [γ-32P ]ATP (6000 Ci/mmol; New England Nuclear) in the provided reaction buffer.

    Plasmid Preparation:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: PCR product was digested by Bam HI and Kpn I, cloned into pGTP_FZ301 previously digested by Bam HI and Kpn I, and ligation was used to transform MG1363. pGTP_FZ301_NucB plasmid extracted from a chloramphenicol resistant clone was verified by digestion and sequencing. .. Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations.

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: .. To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. A pair of scarlet- targeting oligonucleotides were then annealed and ligated into the linearized pDR274 vector using a ligation mix (TaKaRa Bio, Shiga, Japan).

    Article Title: Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase
    Article Snippet: Plasmid pGST‐amylosucrase (pGST‐AS) derived from the pGEX‐6P‐3 (GE Healthcare Biosciences, Piscataway, U.S.A) encoding GST fused to N. polysaccharea amylosucrase, was used for the construction of semi‐rational libraries. .. Dpn I, Eco RI HF and Not I HF restriction enzymes, T4 DNA ligase and Antartic Phosphatase were purchased from New England Biolabs (Beverly, MA, U.S.A.).

    Article Title: VirD - A Virion Display Array For Profiling Functional Membrane Proteins
    Article Snippet: Plasmid pKΔ4B was derived by Cai et al. following engineering of linker-insertion mutants in the glycoprotein B gene. .. DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame . pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences ( ).

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: .. For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette. .. In parallel, the AprR cassette of pIJ773 was amplified using primers Apr1 and Apr2, digested with XbaI and ligated separately with the linearized plasmids described above using T4 ligase (Thermo Scientific, USA).

    Article Title: A Modern Mode of Activation for Nucleic Acid Enzymes
    Article Snippet: Briefly, transcriptions were performed using purified T7 RNA polymerase (10 µg) in the presence of RNA Guard (24 U, Amersham Biosciences) and pyrophosphatase (0.01 U, Roche Diagnostics) in a buffer containing 80 mM HEPES-KOH, pH 7.5, 24 mM MgCl2 , 2 mM spermidine, 40 mM DTT and either linearized plasmid DNA (5 µg) or PCR product (100 pmol) as template. .. In order to produce 5′-labelled RNA substrates, transcripts were dephosphorylated by adding 1 U of antartic phosphatase (New England Biolab) to 20 pmol of RNA and incubating for 30 min at 37°C in a final volume of 10 µL containing 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA and 40 U of RNAGuard (Amersham Biosciences).

    Selection:

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: Since studied K. pneumoniae ST258 is resistant to ampicillin, the selection marker of both vector and clone was replaced by the apramycin resistance cassette of pIJ773. .. For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette.

    In Vitro:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. To synthesize gRNAs in vitro , gRNA synthesis vectors were digested by Dra I (NEW ENGLAND Biolabs, Connecticut, USA) and purified by phenol/chloroform extraction.

    Ethanol Precipitation:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA). .. Dra I-digested DNA fragments were used as templates for in vitro transcription with mMessage mMachine T7 Kit (Life Technologies, California, USA), followed by column purification with miniQuick Spin RNA columns (Roche diagnostics GmbH, Mannheim, Germany), phenol/chloroform extraction, ethanol precipitation, and dissolution in DNase/RNase-free water (Life Technologies, California, USA).

    Knock-Out:

    Article Title: Generation of white-eyed Daphnia magna mutants lacking scarlet function
    Article Snippet: Paragraph title: Knockout of Scarlet by CRISPR/Cas9 system ... To generate the gRNA expression vectors, the plasmid pDR274 (Addgene plasmid 42250, [ ] was digested with Bsa I (NEW ENGLAND Biolabs, Connecticut, USA), followed by dephosphorylation with Antartic Phosphatase (NEW ENGLAND Biolabs, Connecticut, USA).

    DNA Purification:

    Article Title: Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor
    Article Snippet: Restriction enzymes, T4 DNA ligase and Antartic phosphatase (New england Biolabs, Ipswich, MA), high fidelity Phusion™ DNA polymerase (Finnzymes, Espoo, Finland) were used according to recommendations. .. DNA purification kits were purchased from Macherey-Nagel (Düren, Deutschland).

    Marker:

    Article Title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae
    Article Snippet: Since studied K. pneumoniae ST258 is resistant to ampicillin, the selection marker of both vector and clone was replaced by the apramycin resistance cassette of pIJ773. .. For this, both plasmids pBAD-HisA and pBAD-KpndedA were amplified using primers Bad1 and Bad2 , digested with XbaI and dephosphorylated with Antartic phosphatase (New England Biolabs, USA) resulting in the linearized plasmid minus the AmpR cassette.

    Lysis:

    Article Title: From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors
    Article Snippet: Binding of Radioactive Aptamers on Adherent Cells First, 2′F-Py RNAs were dephosphorylated at the 5′-end using antartic phosphatase (New England Biolabs) before 5′-[32 P] labeling (3×103 Bq.pmole−1 ) using T4 kinase. .. After five washings with 500 µL of culture medium, bound sequences were recovered in 200 µL of lysis buffer (50 mM Hepes PH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X100, 2 mM MgCl2 and 5 mM EGTA) and the radioactivity counted.

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    New England Biolabs vector pil253
    Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), <t>pIL253</t> (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.
    Vector Pil253, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector pil253/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vector pil253 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    99
    New England Biolabs antartic phosphatase
    Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), <t>pIL253</t> (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.
    Antartic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antartic phosphatase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antartic phosphatase - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    Image Search Results


    Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.

    Journal: Microbiology

    Article Title: Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract

    doi: 10.1099/mic.0.000515

    Figure Lengend Snippet: Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.

    Article Snippet: A 17 438 bp sequence containing the novel lantibiotic cluster was restricted from the identified pJAZZ-OC clone with ClaI and PstI (NEB) and then ligated into vector pIL253 [MspI, PstI restricted and dephosphorylated (Antarctic Phosphatase, NEB)] using Fastlink DNA ligase (Epicentre) to create p nso.

    Techniques: Western Blot, Hybridization, Marker