m2403s  (New England Biolabs)


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    Name:
    Pyrophosphatase inorganic yeast
    Description:
    Pyrophosphatase inorganic yeast 50 units
    Catalog Number:
    m2403l
    Price:
    273
    Size:
    50 units
    Category:
    Other Enzymes
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    Structured Review

    New England Biolabs m2403s
    Pyrophosphatase inorganic yeast
    Pyrophosphatase inorganic yeast 50 units
    https://www.bioz.com/result/m2403s/product/New England Biolabs
    Average 95 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    m2403s - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: Briefly, the pMCSG7 vector was modified to include the DNA sequence 5′-TAA TAC GAC TCA CTA TAG GAT TTT TTC AGA ATC GAA AGG TTC AAA GTA CAA ATA AGC ATA TAA GGA AAA GAG AGA ATG GGA TCC-3′ using standard ligation-independent cloning techniques. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Amplification:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM). .. Following the amplification, the reaction product was used as the template for another 16S rRNA PCR to determine genomic DNA contamination.

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA). .. The mixture was incubated at 37 °C with gentle rotation on a rotator for 15–17 h. Second RT and PCR amplification: The IVT products attached to the streptavidin beads through the biotin-RT primer were washed once with 0.1× binding buffer (0.5 mM Tris-HCl, pH 7.5; 0.05 mM EDTA; 0.1 M NaCl) on a magnetic separation rack.

    Whole Genome Amplification:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Synthesized:

    Article Title: A versatile polyacrylamide gel electrophoresis based sulfotransferase assay
    Article Snippet: Methods PAP35 S was synthesized from carrier free Na2 35 SO4 (43 Ci/mg, from American Radiolabeled Chemicals, Inc.), ATP and phosphoenol pyruvate using ATP sulfurylase, inorganic pyrophosphatase, pyruvate kinase and APS kinase, and purified as previously described [ , ]. .. ATP sulfurylase and inorganic pyrophosphatase were from New England Biolabs.

    Article Title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts
    Article Snippet: Nucleoside analogues BIM and Benzi ( , ), and triphosphates BIM TP and Benzi TP were synthesized as described previously ( ). .. Pyrophosphatase (Inorganic, Escherichia coli ) was purchased from New England Biolabs.

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Blocking Assay:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Knock-In:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Paragraph title: Detection of knock-in blunt ligation and homologous recombination ... Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

    Incubation:

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: The reactions were incubated overnight at 37 °C, following DNase inactivation at 70 °C for 30 min, and chilled on ice as previously described ( ). .. Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM).

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: Cas9 (M0386S, NEB, Massachusetts, USA) and 5′ adapter-sgRNA were mixed at an equimolar ratio in 1× Cas9 reaction buffer in an appropriate volume (e.g., 10 μl Cas9-sgRNA mix included 1× NEB buffer, 2 pmol Cas9, 60 ng 5′ adapter-sgRNA and water) and incubated at 37 °C for 15 min. Then, 0.2 pmol of preincubated Cas9/sgRNA mixture (1 μl of Cas9/sgRNA mixture prepared above) was added to the RT reaction and incubated at 37 °C for 30 min, followed by the addition of 25 U of RNase If (NEB, Massachusetts, USA) and an incubation at 37 °C for 30 min. On-bead IVT: A T7-promoter oligo (10 pmol) was added to each tube, and the mixture was heated at 95 °C for 5 min and slowly cooled to room temperature. .. IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA).

    Article Title: Murine Norovirus: Propagation, Quantification and Genetic Manipulation
    Article Snippet: ◦ 10 µl 0.4M Tris pH 8.0 ◦ 10 µl 320mM Magnesium acetate ◦ 10 µl 400mM DTT ◦ 10 µl 20mM spermidine ◦ 7.5 µl 100mM ATP ◦ 7.5 µl 100mM GTP ◦ 7.5 µl 100mM CTP ◦ 7.5 µl 100mM UTP ◦ 2 µl Pyrophosphatase (New England Biolabs, M2403L) ◦ 5 µl 40 U/µl RNaseOUT (Life Technologies, 10777-019) ◦ 10 µl PCR product ( > 200ng) ◦ 10 µl T7 RNA polymerase (Promega, P2074) ◦ Nuclease-free water to 100 µl. .. 6 Add 2 µl DNase I and continue the incubation at 37°C water bath for another30 minutes.

    Article Title:
    Article Snippet: Briefly, the indicated conditions were pipetted into BSA-precoated tubes containing an estimated 30 nM SYNAC and 0.05 U inorganic pyrophosphatase (from Escherichia coli; New England Biolabs, Ipswich, MA). .. Tubes were incubated at 30°C for 30 minutes before being divided into 20 μ l aliquots and halted with an equal addition of Kinase-Glo Max reagent.

    Activity Assay:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: In vitro tests of cap state selection For tests of CoPRO treatments, radiolabeled RNA was made by incorporating P into the body of the RNA during transcription, using home-made T7 RNA polymerase and buffer (30 mM HEPES pH 7.8, 80mM Potassium Glutamate, 15mM MgAc, 0.25 mM EDTA, 5 mM DTT, 0.05% Tween-20, 2 mM Spermidine, 2.5 mM ATP, GTP, and UTP, and 0.25 mM cold CTP), with YIPP (NEB cat. M2403S) and Superase-In (ThermoFisher cat. AM2696) added as per the manufacturer’s protocol. .. The ability of different series of treatments to selectively reduce capped and uncapped RNA to monophosphate was assessed in two ways: first by using Terminator degradation as readout as it requires a 5’ monophosphate for its exonuclease activity just as adapter ligation requires a 5’ monophosphate in library preparation , and second by using ligation of the 5’ adapter from a standard PRO-seq as the readout ( ).

    Article Title:
    Article Snippet: Paragraph title: Cyclase Activity. ... Briefly, the indicated conditions were pipetted into BSA-precoated tubes containing an estimated 30 nM SYNAC and 0.05 U inorganic pyrophosphatase (from Escherichia coli; New England Biolabs, Ipswich, MA).

    Expressing:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. For mRNA MRFF, the corresponding coding sequence (underlined) with a T7 promoter sequence (GGG AAT TCG AAA TAG AAG TCT TCT TTT TGG A AAA ATT TAA AAG TTA ATA AGG ATA CAT ACT ATG CGT TTC TTC CGT TTC TTC CGT AAA TTC CGT GTG CGT TTT TTC AAA TTT GTG TTC CGT TAA CGC GTC TGC AGG CAT GCA AGC TAA AAA AAA AAA AAA AAA AAA AAA AAA GCT T), purchased from IDT, was ligated into plasmid pTZ18R for expression and purification. tRNAVal2B and mutants were prepared by in vitro transcription as previously described.

    Modification:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Modified cells were subjected to FACS four to seven days after nucleofection. .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA. .. The reaction was then purified by acidic phenol:chloroform extraction and ethanol precipitated at −80°C for 16 h. The RNA pellet was further purified using 70% ethanol and air dried before resuspending in 100 µl of 10 mM sodium cacodylate, pH 6.5.

    Activated Clotting Time Assay:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Ligation:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Paragraph title: Detection of knock-in blunt ligation and homologous recombination ... Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: In vitro tests of cap state selection For tests of CoPRO treatments, radiolabeled RNA was made by incorporating P into the body of the RNA during transcription, using home-made T7 RNA polymerase and buffer (30 mM HEPES pH 7.8, 80mM Potassium Glutamate, 15mM MgAc, 0.25 mM EDTA, 5 mM DTT, 0.05% Tween-20, 2 mM Spermidine, 2.5 mM ATP, GTP, and UTP, and 0.25 mM cold CTP), with YIPP (NEB cat. M2403S) and Superase-In (ThermoFisher cat. AM2696) added as per the manufacturer’s protocol. .. The ability of different series of treatments to selectively reduce capped and uncapped RNA to monophosphate was assessed in two ways: first by using Terminator degradation as readout as it requires a 5’ monophosphate for its exonuclease activity just as adapter ligation requires a 5’ monophosphate in library preparation , and second by using ligation of the 5’ adapter from a standard PRO-seq as the readout ( ).

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: Briefly, the pMCSG7 vector was modified to include the DNA sequence 5′-TAA TAC GAC TCA CTA TAG GAT TTT TTC AGA ATC GAA AGG TTC AAA GTA CAA ATA AGC ATA TAA GGA AAA GAG AGA ATG GGA TCC-3′ using standard ligation-independent cloning techniques. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Mutagenesis:

    Article Title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts
    Article Snippet: KTqM747K mutant DNA polymerase was kindly provided by myPOLS Biotec GmbH. .. Pyrophosphatase (Inorganic, Escherichia coli ) was purchased from New England Biolabs.

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Polymerase Chain Reaction:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: When 16S rRNA PCR product was visible on an electrophoretic DNA gel, another overnight digestion reaction was performed until 16S rRNA PCR product could no longer be visualized. .. Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM).

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: A 50 μL reaction composed of 20 μL DNA, 24 μL MilliQ water, 1 μL ATP, 2.5 μL reaction buffer, and 2.5 μL plasmid-safe DNase was incubated at 37°C overnight and deactivated at 70°C for 30 min. Amplification of the 16S rRNA genes by PCR as previously described was performed to ensure the ratio of plasmid:chromosomal DNA is was reversed in the sample, i.e., high plasmid to low chromosomal DNA ratio. .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA). .. The mixture was incubated at 37 °C with gentle rotation on a rotator for 15–17 h. Second RT and PCR amplification: The IVT products attached to the streptavidin beads through the biotin-RT primer were washed once with 0.1× binding buffer (0.5 mM Tris-HCl, pH 7.5; 0.05 mM EDTA; 0.1 M NaCl) on a magnetic separation rack.

    Article Title: Murine Norovirus: Propagation, Quantification and Genetic Manipulation
    Article Snippet: .. ◦ 10 µl 0.4M Tris pH 8.0 ◦ 10 µl 320mM Magnesium acetate ◦ 10 µl 400mM DTT ◦ 10 µl 20mM spermidine ◦ 7.5 µl 100mM ATP ◦ 7.5 µl 100mM GTP ◦ 7.5 µl 100mM CTP ◦ 7.5 µl 100mM UTP ◦ 2 µl Pyrophosphatase (New England Biolabs, M2403L) ◦ 5 µl 40 U/µl RNaseOUT (Life Technologies, 10777-019) ◦ 10 µl PCR product ( > 200ng) ◦ 10 µl T7 RNA polymerase (Promega, P2074) ◦ Nuclease-free water to 100 µl. .. 5 Run 2 µl on denaturing 5% PAGE (Biorad mini protean gel or similar) to check the yield and purity of the RNA ( ).

    Recombinant:

    Article Title: A versatile polyacrylamide gel electrophoresis based sulfotransferase assay
    Article Snippet: ATP sulfurylase and inorganic pyrophosphatase were from New England Biolabs. .. Recombinant syndecan-4 was from R & D Systems.

    Cellular Antioxidant Activity Assay:

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: Briefly, the pMCSG7 vector was modified to include the DNA sequence 5′-TAA TAC GAC TCA CTA TAG GAT TTT TTC AGA ATC GAA AGG TTC AAA GTA CAA ATA AGC ATA TAA GGA AAA GAG AGA ATG GGA TCC-3′ using standard ligation-independent cloning techniques. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Marker:

    Article Title: Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex
    Article Snippet: .. The marker m7G*pppA was prepared as above, except that inorganic pyrophosphatase was replaced by 0.2 mM SAM. ..

    Multiple Displacement Amplification:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: Paragraph title: Multiple displacement amplification ... Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Isolation:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Labeling:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. L11 labeling with Cy3 and the reconstitution of 70S ribosomes containing L11Cy3 (70SCy3 ) were performed as described.

    Purification:

    Article Title: A versatile polyacrylamide gel electrophoresis based sulfotransferase assay
    Article Snippet: Methods PAP35 S was synthesized from carrier free Na2 35 SO4 (43 Ci/mg, from American Radiolabeled Chemicals, Inc.), ATP and phosphoenol pyruvate using ATP sulfurylase, inorganic pyrophosphatase, pyruvate kinase and APS kinase, and purified as previously described [ , ]. .. ATP sulfurylase and inorganic pyrophosphatase were from New England Biolabs.

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: Plasmid DNA was amplified by adding 1 μL of 10 μM Exo-Resistant Random Primer (Thermo Scientific), 2 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs) and 8.2 μL of MilliQ water to 5 μL of the purified treated DNA. .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: RNA samples and generation of the 5′-UTR of hfq mRNA Oligoribonucleotides were purchased from IDT (Coralville, IA) or Oligos Etc (Wilsonville, OR) and used without further purification. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Article Title: T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures
    Article Snippet: Non-modified DNA was ordered PAGE purified. .. T7 RNAP was purchased from Cellscript (200 U/μl, Catalog # C-T7300K), SP6 RNAP from ThermoFisher Scientific (100 U/μl, Catalog # EP0133), yeast inorganic pyrophosphatase from New England Biolabs (NEB) (0.1 U/μl, Catalog # M2403S) and DNase I from NEB (2 U/μl, Catalog # M0303S).

    Sequencing:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: Briefly, the pMCSG7 vector was modified to include the DNA sequence 5′-TAA TAC GAC TCA CTA TAG GAT TTT TTC AGA ATC GAA AGG TTC AAA GTA CAA ATA AGC ATA TAA GGA AAA GAG AGA ATG GGA TCC-3′ using standard ligation-independent cloning techniques. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: The RT reaction was performed at 44 °C for 1 h. Cas9/sgRNA treatment: An sgRNA with the indicated sequence (Fig. ) was produced by IVT using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). .. IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA).

    Positron Emission Tomography:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Polyacrylamide Gel Electrophoresis:

    Article Title: T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures
    Article Snippet: Non-modified DNA was ordered PAGE purified. .. T7 RNAP was purchased from Cellscript (200 U/μl, Catalog # C-T7300K), SP6 RNAP from ThermoFisher Scientific (100 U/μl, Catalog # EP0133), yeast inorganic pyrophosphatase from New England Biolabs (NEB) (0.1 U/μl, Catalog # M2403S) and DNase I from NEB (2 U/μl, Catalog # M0303S).

    Article Title: Murine Norovirus: Propagation, Quantification and Genetic Manipulation
    Article Snippet: ◦ 10 µl 0.4M Tris pH 8.0 ◦ 10 µl 320mM Magnesium acetate ◦ 10 µl 400mM DTT ◦ 10 µl 20mM spermidine ◦ 7.5 µl 100mM ATP ◦ 7.5 µl 100mM GTP ◦ 7.5 µl 100mM CTP ◦ 7.5 µl 100mM UTP ◦ 2 µl Pyrophosphatase (New England Biolabs, M2403L) ◦ 5 µl 40 U/µl RNaseOUT (Life Technologies, 10777-019) ◦ 10 µl PCR product ( > 200ng) ◦ 10 µl T7 RNA polymerase (Promega, P2074) ◦ Nuclease-free water to 100 µl. .. 5 Run 2 µl on denaturing 5% PAGE (Biorad mini protean gel or similar) to check the yield and purity of the RNA ( ).

    FACS:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Modified cells were subjected to FACS four to seven days after nucleofection. .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

    IA:

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: RNA samples and generation of the 5′-UTR of hfq mRNA Oligoribonucleotides were purchased from IDT (Coralville, IA) or Oligos Etc (Wilsonville, OR) and used without further purification. .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA.

    Agarose Gel Electrophoresis:

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM). .. Reactions were incubated at 30 °C for 16 h. Finally, 3 μL from each reaction was loaded onto a 1% agarose gel with ethidium bromide staining for analysis.

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. .. Plasmids were visualized on a 1% agarose gel stained with GelRed run at 70 V for 60 mins.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. For mRNA MRFF, the corresponding coding sequence (underlined) with a T7 promoter sequence (GGG AAT TCG AAA TAG AAG TCT TCT TTT TGG A AAA ATT TAA AAG TTA ATA AGG ATA CAT ACT ATG CGT TTC TTC CGT TTC TTC CGT AAA TTC CGT GTG CGT TTT TTC AAA TTT GTG TTC CGT TAA CGC GTC TGC AGG CAT GCA AGC TAA AAA AAA AAA AAA AAA AAA AAA AAA GCT T), purchased from IDT, was ligated into plasmid pTZ18R for expression and purification. tRNAVal2B and mutants were prepared by in vitro transcription as previously described.

    Plasmid Preparation:

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: Reactions contained: 5 μL of the plasmid DNA as template, 1.5 μL of 10 μM exonuclease-resistant random hexamers (Fermentas), 3 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs), and 15.7 μL double-distilled water. .. Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM).

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. ..

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: .. Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Article Title: Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching
    Article Snippet: .. To prepare RNA, the modified vector was midiprepped, phenol:chloroform extracted and 8–20 µg of clean DNA vector was digested using BamH1-HF (NEB, Ipswich, MA) at 37°C for 16 h. The T7 RNA polymerase reaction [100 µl of 10× Buffer (0.5 M Tris base, pH 7.5, 0.25 M MgCl2 , 0.05 M EDTA), 40 µl of 50 mM spermidine, 200 µl of rNTP mix (20 mM/rNTP), 30 µl of 100 units/ml inorganic pyrophosphatase (NEB), 80 µl of 1 mg/ml T7 RNA polymerase, 60 µl of 8–20 µg/ml digested DNA vector, 440 µl of dH2 O) was carried out at 37°C for 16 h and quenched with 2 ml of 0.45 M EDTA. .. The reaction was then purified by acidic phenol:chloroform extraction and ethanol precipitated at −80°C for 16 h. The RNA pellet was further purified using 70% ethanol and air dried before resuspending in 100 µl of 10 mM sodium cacodylate, pH 6.5.

    Binding Assay:

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA). .. The mixture was incubated at 37 °C with gentle rotation on a rotator for 15–17 h. Second RT and PCR amplification: The IVT products attached to the streptavidin beads through the biotin-RT primer were washed once with 0.1× binding buffer (0.5 mM Tris-HCl, pH 7.5; 0.05 mM EDTA; 0.1 M NaCl) on a magnetic separation rack.

    Sample Prep:

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: Paragraph title: Sample Preparation. ... Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM).

    In Vitro:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: .. In vitro tests of cap state selection For tests of CoPRO treatments, radiolabeled RNA was made by incorporating P into the body of the RNA during transcription, using home-made T7 RNA polymerase and buffer (30 mM HEPES pH 7.8, 80mM Potassium Glutamate, 15mM MgAc, 0.25 mM EDTA, 5 mM DTT, 0.05% Tween-20, 2 mM Spermidine, 2.5 mM ATP, GTP, and UTP, and 0.25 mM cold CTP), with YIPP (NEB cat. M2403S) and Superase-In (ThermoFisher cat. AM2696) added as per the manufacturer’s protocol. .. Capped RNA was made by vaccinia capping (NEB cat. M2080S), but without subsequent uncapped clean-up: we estimate that capping was 80% complete (data not shown).

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. For mRNA MRFF, the corresponding coding sequence (underlined) with a T7 promoter sequence (GGG AAT TCG AAA TAG AAG TCT TCT TTT TGG A AAA ATT TAA AAG TTA ATA AGG ATA CAT ACT ATG CGT TTC TTC CGT TTC TTC CGT AAA TTC CGT GTG CGT TTT TTC AAA TTT GTG TTC CGT TAA CGC GTC TGC AGG CAT GCA AGC TAA AAA AAA AAA AAA AAA AAA AAA AAA GCT T), purchased from IDT, was ligated into plasmid pTZ18R for expression and purification. tRNAVal2B and mutants were prepared by in vitro transcription as previously described.

    Article Title: Murine Norovirus: Propagation, Quantification and Genetic Manipulation
    Article Snippet: Paragraph title: Generate RNA using in vitro transcription ... ◦ 10 µl 0.4M Tris pH 8.0 ◦ 10 µl 320mM Magnesium acetate ◦ 10 µl 400mM DTT ◦ 10 µl 20mM spermidine ◦ 7.5 µl 100mM ATP ◦ 7.5 µl 100mM GTP ◦ 7.5 µl 100mM CTP ◦ 7.5 µl 100mM UTP ◦ 2 µl Pyrophosphatase (New England Biolabs, M2403L) ◦ 5 µl 40 U/µl RNaseOUT (Life Technologies, 10777-019) ◦ 10 µl PCR product ( > 200ng) ◦ 10 µl T7 RNA polymerase (Promega, P2074) ◦ Nuclease-free water to 100 µl.

    Selection:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: .. In vitro tests of cap state selection For tests of CoPRO treatments, radiolabeled RNA was made by incorporating P into the body of the RNA during transcription, using home-made T7 RNA polymerase and buffer (30 mM HEPES pH 7.8, 80mM Potassium Glutamate, 15mM MgAc, 0.25 mM EDTA, 5 mM DTT, 0.05% Tween-20, 2 mM Spermidine, 2.5 mM ATP, GTP, and UTP, and 0.25 mM cold CTP), with YIPP (NEB cat. M2403S) and Superase-In (ThermoFisher cat. AM2696) added as per the manufacturer’s protocol. .. Capped RNA was made by vaccinia capping (NEB cat. M2080S), but without subsequent uncapped clean-up: we estimate that capping was 80% complete (data not shown).

    Column Chromatography:

    Article Title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts
    Article Snippet: Pyrophosphatase (Inorganic, Escherichia coli ) was purchased from New England Biolabs. .. Flash column chromatography was performed on a Biotage system with pre-packed Flash+ KP-SiO2 cartridges.

    Nuclear Magnetic Resonance:

    Article Title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts
    Article Snippet: Pyrophosphatase (Inorganic, Escherichia coli ) was purchased from New England Biolabs. .. 1 H, 13 C and 31 P NMR spectra were recorded on a Bruker Biospin 400 MHz NMR instrument, and chemical shifts are reported in parts per million (ppm, δ ) relative to the chemical shift of the respective NMR solvent.

    Produced:

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: The RT reaction was performed at 44 °C for 1 h. Cas9/sgRNA treatment: An sgRNA with the indicated sequence (Fig. ) was produced by IVT using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). .. IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA).

    Concentration Assay:

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: .. IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA). .. The mixture was incubated at 37 °C with gentle rotation on a rotator for 15–17 h. Second RT and PCR amplification: The IVT products attached to the streptavidin beads through the biotin-RT primer were washed once with 0.1× binding buffer (0.5 mM Tris-HCl, pH 7.5; 0.05 mM EDTA; 0.1 M NaCl) on a magnetic separation rack.

    Thin Layer Chromatography:

    Article Title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts
    Article Snippet: Pyrophosphatase (Inorganic, Escherichia coli ) was purchased from New England Biolabs. .. Silica gel 60 F254 plates with aluminum backing were used for thin layer chromatography.

    cDNA Library Assay:

    Article Title: Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes
    Article Snippet: Paragraph title: CAS-seq cDNA library preparation ... IVT was performed with cDNA on beads in a 20-μl reaction volume containing 1× Transcription Optimized buffer (Promega, Wisconsin, USA), 4 mM of each rNTP, 14.5 mM MgCl2 (Sigma, USA), 2.5 pmol of the T7-promoter oligo, 10 mM DTT, 40 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), 0.08 U of inorganic pyrophosphatase (NEB, Massachusetts, USA), and 16 U of high concentration T7 RNA Polymerase (80 U/μl, Promega, Wisconsin, USA).

    CTG Assay:

    Article Title: Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation
    Article Snippet: Materials The following materials were purchased from the indicated suppliers: IPTG, GDP, GTP, GDPNP [guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate], TCEP [tris(2-carboxyethyl)phosphine hydrochloride], streptomycin, paromomycin, kirromycin, and poly(U), bulk E.coli tRNA, amino acids (Sigma); Talon Superflow Metal Affinity Resin (Clontech); plasmid pTZ18R (Amersham); QSY9 maleimide, dNTP, and Top 10 competent cells (Invitrogen); Cy3 maleimide, Cy5 maleimide, NAP-5 column, pGEX vector, MonoQ column (GE Healthcare); pET-15b vector (Novagen); QuikChange site-directed mutagenesis kit (Stratagene); phosphoenolpyruvate (PEP), pyruvate kinase (PK), and RNase A (Roche); proflavin (MB Biomedicals); inorganic pyrophosphatase, Phusion DNA polymerase and DpnI (New England Biolabs); PCR purification kit (QIAGEN); Amicon Ultra ultrafiltration units and nitrocellulose filters (Millipore); GF/C filters (Whatman); E. coli tRNAfMet and yeast tRNAPhe (Chemical Block, Moscow). .. Tightly coupled wild-type 70S ribosomes from E. coli MRE600 cells, mutant 70S ribosomes lacking L11 from E. coli AM77 cells, cloned E. coli His-tagged proteins (IF1-3, L11(C38S/S87C), and EF-G), and mRNA 022 and 022CUC were prepared and purified as previously reported. mRNA MVYF was prepared by mutation of the mRNA 022 sequence via PCR, using the following primers: forward, 5′-TTA ACT TTT AAA TTT TTG AAT TCC CTA TAG TGA GTC GTA TTA AAT TC-3′; over-reverse, 5′-CAG GTA TAC ATA CTA TGG TCT ACT TTA CTA CGA TCT TCT TCA CTT AAC GCG TCT GCA GGC ATG-3′; reverse, 5′-CTT CAC TTA ACG CGT CTG CAG GCA TG-3′; over-forward, 5′-AAG ATC GTA GTA AAG TAG ACC ATA GTA TGT ATA CCT GTT AAC TTT TAA ATT TTT GAA TTC CCT ATA GTG AGT CGT ATT AAA TTC-3′.

    Staining:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Harvested cells were resuspended in DPBS (Life Technologies) with 2% AlbuMAX I (Life Technologies), 2 mM EDTA (Life Technologies), NucBlue Fixed Cell Stain ReadyProbes reagent, and 10 μM ROCK inhibitor Y-27632, and subsequently filtered to remove clumps and debris. .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

    Article Title: Insights into the bovine rumen plasmidome
    Article Snippet: Following 5 min on ice, 1.6 μL phi29 DNA polymerase was added along with 0.2 μL pyrophosphatase, inorganic (yeast) (New England Biolabs) and 3 μL dNTPs (10 mM). .. Reactions were incubated at 30 °C for 16 h. Finally, 3 μL from each reaction was loaded onto a 1% agarose gel with ethidium bromide staining for analysis.

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells. .. Plasmids were visualized on a 1% agarose gel stained with GelRed run at 70 V for 60 mins.

    Variant Assay:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: .. Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB). .. PCR-amplified products were purified with the MinElute PCR Purification kit (Qiagen), diluted to 100 μl with Buffer EB (Qiagen), and used for further genome-cassette junction PCR amplifications.

    Homologous Recombination:

    Article Title: In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
    Article Snippet: Paragraph title: Detection of knock-in blunt ligation and homologous recombination ... Genomic DNA was isolated from sorted cells using the ZymoBead Genomic DNA kit, (Zymo Research, Irvine, CA). gDNA was then subjected to targeted amplification of the H11 locus via PCR amplification with Q5 high-fidelity polymerase or multiple displacement amplification (MDA; a variant of whole genome amplification) following , with the addition of inorganic (yeast) pyrophosphatase (NEB).

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    New England Biolabs yeast inorganic pyrophosphatase
    HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, <t>pyrophosphatase-treated</t> (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.
    Yeast Inorganic Pyrophosphatase, 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
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    HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase

    doi: 10.1073/pnas.1214924110

    Figure Lengend Snippet: HCV polymerase (NS5B) catalyzes NTP-mediated excision of a 3′-terminal CMP from a nascent RNA primer. ( A ) Procedure to obtain a purified NS5B–10/20-mer elongation complex with α- 32 P-CMP (indicated as C ) at the 3′-end of the 10-mer primer. The sequence of the RNA template (20-mer) used in this study is shown. ( B ) Sequencing gel image shows excision of the α- 32 P-CMP from 10-mer RNA substrate by PP i or NTPs and generation of CTP or Np 4 C dinucleoside tetraphosphates. For each reaction, the purified NS5B–10/20-mer elongation complex (0.3 μM) was incubated for 7 min with 1 mM PP i or NTP, pyrophosphatase-treated (+) or untreated (−). Lane 1: NS5B–10/20-mer elongation complex before purification. rxn, reaction. Lane 2: purified elongation complex. As a position reference, dinucleoside tetraphosphates Ap 4 C, Up 4 C, Cp 4 C, and Gp 4 C synthesized by firefly luciferase were loaded in lanes 7, 10, 13, and 16, respectively, and their positions are indicated by arrows.

    Article Snippet: In a 50-μL reaction, 20 μCi [α-32 P]CTP and one of the NTPs (ATP, GTP, UTP, or CTP) at 2 mM were incubated for 24 h at 30 °C with 25 μg of firefly luciferase (Sigma–Aldrich) in a reaction buffer containing 50 mM Hepes (pH 7.4), 5 mM MgCl2 , 1 mg/mL BSA, 0.05 units of yeast inorganic pyrophosphatase (PPase; New England Biolabs), and 0.1 mM D-Luciferin (Sigma–Aldrich).

    Techniques: Purification, Sequencing, Incubation, Synthesized, Luciferase

    MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: MicroRNA synthesized by rolling circle transcription (RCT) site-specific disconnection (SSD) with various concentrations of circular cDNA template . ( a ) Time course of RCT in the absence of inorganic pyrophosphatase (IPP). ( b ) Effect of IPP on the amount of RNA products. Lane L, 25 nt RNA marker; Lane S, standard sample of chemically synthesized mir-16 (1 μl, 100 μmol/l). In ( b ), RCT was processed for 4 hours, with 0.1 μmol/l Aid-DNA-16-1 and 1.0 U/ml IPP.

    Article Snippet: Inorganic pyrophosphatase (0.02 U) purchased from New England Biolabs, was applied to selected RCT reactions.

    Techniques: Synthesized, Marker