agei hf  (New England Biolabs)


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    Name:
    AgeI HF
    Description:
    AgeI HF 1 500 units
    Catalog Number:
    r3552l
    Price:
    298
    Size:
    1 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs agei hf
    AgeI HF
    AgeI HF 1 500 units
    https://www.bioz.com/result/agei hf/product/New England Biolabs
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    agei hf - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli"

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1018

    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.
    Figure Legend Snippet: RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Techniques Used: Purification, In Vitro, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Ligation, Sequencing

    Related Articles

    Clone Assay:

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: Cib81 was PCR amplified to add on BsaI and linkers (Ala–Ser and Ser–Ala–Gly), preceding and following the domain insertion) sites complementary to MuA-BsaI transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water.

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: The recombinase genes were cloned into pNPK-plasmids as previously described . .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To facilitate cloning of the pooled PCR products, the PCR primers were designed to have an additional 15 nt recombination arms at 5′ end of each forward primer (TAGAGCATGCACCGG) and reverse primer (GGCCGAATTCGTCGA).

    Article Title: Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling
    Article Snippet: .. Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs (Ipswich, MA, USA). pLKO.1‐TRC cloning vector was a gift from David Root (Addgene plasmid # 10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10878). .. Cell Line and Cell Culture Human GCTB Hs 737.T (ATCC, USA) were grown in Dulbecco's modified eagle medium plus 10% FBS, l00 μg/mL streptomycin and penicillin.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: In short, libraries for in vivo screening were cloned by homologues recombination in yeast. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: Domains (PDZ (GI: 404931, https://www.ncbi.nlm.nih.gov/protein/404931 ), Cib81 , GSAGx2 , and GSAGx3 ) for insertions were ordered as gBlocks (IDT DNA), and PCR amplified to add on BsaI and linkers (Ala-Ser and Ser-Ala-Gly using primers 77–84 (Supplementary Table ), preceding and following the domain insertion), sites complementary to MuA-BsaI-transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water.

    Centrifugation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: Upon centrifugation of the reaction mixture over a sucrose cushion to collect ribosomes, RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction ( ). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    Amplification:

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: Cib81 was PCR amplified to add on BsaI and linkers (Ala–Ser and Ser–Ala–Gly), preceding and following the domain insertion) sites complementary to MuA-BsaI transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water.

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: The puromycin-resistance gene was amplified from pIRESpuro3-mRedNLS using primers that contain a recombination target site each as well as AgeI restriction sites. .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene.

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: FPR1 cDNA was amplified from a cDNA plasmid template (1 μl of glycerol bacterial stock) using a PAGE purified forward primer 2A_FPR1_fwd (GCT ACT AAC TTC TCT CTA TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCA GGT CCT ATG GAG ACA AAT TCC TCT CTC CC) and reverse primer FPR1_rev (GCG GAG GCC ACG CGT CTA CTT TGC CTG TAA CTC CAC C). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For that, RT-PCR product of a defined round was amplified with CFX_HR_fwd (5′-CAA GCT ATA CCA AGC ATA CAA TCA ACT CCA AGC TAG ATC TAC CGG TGG GAG ACG CAA CTG AAT GAA-3′) and CFX_HR_rev (5′-CAA GAA TTG GGA CAA CTC CAG TGA AAA GTT CTT CTC CTT TGC TAG CGT GAC GCG ACT AGT TAC GGA-3′) to attach 46 bp overhang for recombination into pWHE601*. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: Domains (PDZ (GI: 404931, https://www.ncbi.nlm.nih.gov/protein/404931 ), Cib81 , GSAGx2 , and GSAGx3 ) for insertions were ordered as gBlocks (IDT DNA), and PCR amplified to add on BsaI and linkers (Ala-Ser and Ser-Ala-Gly using primers 77–84 (Supplementary Table ), preceding and following the domain insertion), sites complementary to MuA-BsaI-transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water.

    Construct:

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: The RFP-based recombination reporter plasmids (pRed) were constructed in a way that site-specific excision of a stop-cassette flanked by co-oriented recombination target sites activates the expression of red fluorescent protein (RFP). .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene.

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: A lentiviral plasmid expressing turbo red fluorescent protein (tRFP) and FPR1 cDNA separated by a 2A sequence (pTripz_RFP_2A_FPR1) was constructed by amplification of a tRFP fragment from a 10 ng pTripz_control plasmid template using forward primer RFP_fwd (CGT TTA GTG AAC CGT CAG ATC GCA CCG GTC GCC ACC ATG AG) and reverse primer RFP_2A_rev (GTC TCC TGC TTG CTT TAA TAG AGA GAA GTT AGT AGC TCC AGA TCC TCT GTG CCC CAG TTT GCT A). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Real-time Polymerase Chain Reaction:

    Article Title: Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling
    Article Snippet: Puromycin, streptomycin and penicillin, Lipofectamine 3000 Transfection Reagent, RT reagent Kit, and quantitative polymerase chain reaction (qPCR) kits were from Thermo Fisher Scientific (Waltham, MA, USA). .. Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs (Ipswich, MA, USA). pLKO.1‐TRC cloning vector was a gift from David Root (Addgene plasmid # 10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10878).

    Incubation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Expressing:

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: The RFP-based recombination reporter plasmids (pRed) were constructed in a way that site-specific excision of a stop-cassette flanked by co-oriented recombination target sites activates the expression of red fluorescent protein (RFP). .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene.

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: Paragraph title: Generation of neuroblastoma cells with a differential expression of FPR1 ... PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. For the first screening round, cells were selected under the fluorescence binocular and checked for GFP expression.

    Modification:

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene. .. The viral recombinase expression constructs used in the cytotoxicity studies were based on pBabe-puro (Addgene plasmid 1764) and modified to contain an internal ribosomal entry site and a GFP reporter gene as previously described .

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: LentiX HEK-293 T cells were purchased from Clonetech (632180, Mountain View, CA, USA) and grown at 37 °C and 5 % CO2 in Dulbecco’s Modified Eagle Medium (11966–025, Gibco™, Paisley, UK), supplemented with 10 % FBS; 100 μg/mL of streptomycin and 100 U/mL of penicillin. .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Transformation Assay:

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: The remaining transformation mix was grown in LB containing kanamycin (50 μg/ml) and chloramphenicol (25 μg/ml). .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. Transformed cells were spread on several SCD-ura plates in a way that assures a moderate colony density which simplifies picking clones.

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: All transformed libraries yielded greater than 105 colonies, so for Kir2.1 (1369 bp) there is > 35x coverage. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water.

    Gel Purification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Countercurrent Chromatography:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: A lentiviral plasmid expressing turbo red fluorescent protein (tRFP) and FPR1 cDNA separated by a 2A sequence (pTripz_RFP_2A_FPR1) was constructed by amplification of a tRFP fragment from a 10 ng pTripz_control plasmid template using forward primer RFP_fwd (CGT TTA GTG AAC CGT CAG ATC GCA CCG GTC GCC ACC ATG AG) and reverse primer RFP_2A_rev (GTC TCC TGC TTG CTT TAA TAG AGA GAA GTT AGT AGC TCC AGA TCC TCT GTG CCC CAG TTT GCT A). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Transfection:

    Article Title: Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling
    Article Snippet: Puromycin, streptomycin and penicillin, Lipofectamine 3000 Transfection Reagent, RT reagent Kit, and quantitative polymerase chain reaction (qPCR) kits were from Thermo Fisher Scientific (Waltham, MA, USA). .. Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs (Ipswich, MA, USA). pLKO.1‐TRC cloning vector was a gift from David Root (Addgene plasmid # 10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10878).

    Ligation:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel. .. A 2 μl ligation mixture was used to transfect XL10 gold ultracompetent cells (200314, NEB) and spread on 100 μM Amp agar plates.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For that, RT-PCR product of a defined round was amplified with CFX_HR_fwd (5′-CAA GCT ATA CCA AGC ATA CAA TCA ACT CCA AGC TAG ATC TAC CGG TGG GAG ACG CAA CTG AAT GAA-3′) and CFX_HR_rev (5′-CAA GAA TTG GGA CAA CTC CAG TGA AAA GTT CTT CTC CTT TGC TAG CGT GAC GCG ACT AGT TAC GGA-3′) to attach 46 bp overhang for recombination into pWHE601*. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Generated:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: Rx2 and actb template plasmids for gfp donor cassette amplification are described in and were generated by GoldenGATE assembly into the pGGDestSC-ATG destination vector (addgene #49322) according to . .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    other:

    Article Title: Targeted removal of epigenetic barriers during transcriptional reprogramming
    Article Snippet: The backbone was digested with AgeI-HF.

    Sequencing:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: A lentiviral plasmid expressing turbo red fluorescent protein (tRFP) and FPR1 cDNA separated by a 2A sequence (pTripz_RFP_2A_FPR1) was constructed by amplification of a tRFP fragment from a 10 ng pTripz_control plasmid template using forward primer RFP_fwd (CGT TTA GTG AAC CGT CAG ATC GCA CCG GTC GCC ACC ATG AG) and reverse primer RFP_2A_rev (GTC TCC TGC TTG CTT TAA TAG AGA GAA GTT AGT AGC TCC AGA TCC TCT GTG CCC CAG TTT GCT A). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Cellular Antioxidant Activity Assay:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: FPR1 cDNA was amplified from a cDNA plasmid template (1 μl of glycerol bacterial stock) using a PAGE purified forward primer 2A_FPR1_fwd (GCT ACT AAC TTC TCT CTA TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCA GGT CCT ATG GAG ACA AAT TCC TCT CTC CC) and reverse primer FPR1_rev (GCG GAG GCC ACG CGT CTA CTT TGC CTG TAA CTC CAC C). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For that, RT-PCR product of a defined round was amplified with CFX_HR_fwd (5′-CAA GCT ATA CCA AGC ATA CAA TCA ACT CCA AGC TAG ATC TAC CGG TGG GAG ACG CAA CTG AAT GAA-3′) and CFX_HR_rev (5′-CAA GAA TTG GGA CAA CTC CAG TGA AAA GTT CTT CTC CTT TGC TAG CGT GAC GCG ACT AGT TAC GGA-3′) to attach 46 bp overhang for recombination into pWHE601*. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Nucleic Acid Electrophoresis:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    In Vivo:

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: Paragraph title: In vivo screening ... Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Fluorescence:

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. For the first screening round, cells were selected under the fluorescence binocular and checked for GFP expression.

    Isolation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Purification:

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: Domain amplicons were gel purified (Zymo Research). .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water.

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: The RT-PCR product was analyzed on 10% PAA gels in TBE buffer, and purified using ‘PCR clean up kit’ (Qiagen, Hilden, Germany). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel. .. 100 ng of plasmid backbone was mixed with the PCR-generated tRFP and FPR1 cDNA fragments in equimolar amounts and ligated using 2X Gibson mix (E2611S, NEB).

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: .. The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To prepare inserts (candidate SNP-containing sequences), we performed PCR to amplify DNA fragments covering all SNPs selected from SNPs-seq.

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: Domain amplicons were gel purified (Zymo Research). .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water.

    Polymerase Chain Reaction:

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: Cib81 was PCR amplified to add on BsaI and linkers (Ala–Ser and Ser–Ala–Gly), preceding and following the domain insertion) sites complementary to MuA-BsaI transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water.

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene. .. The viral recombinase expression constructs used in the cytotoxicity studies were based on pBabe-puro (Addgene plasmid 1764) and modified to contain an internal ribosomal entry site and a GFP reporter gene as previously described .

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: The RT-PCR product was analyzed on 10% PAA gels in TBE buffer, and purified using ‘PCR clean up kit’ (Qiagen, Hilden, Germany). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel. .. 100 ng of plasmid backbone was mixed with the PCR-generated tRFP and FPR1 cDNA fragments in equimolar amounts and ligated using 2X Gibson mix (E2611S, NEB).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To prepare inserts (candidate SNP-containing sequences), we performed PCR to amplify DNA fragments covering all SNPs selected from SNPs-seq.

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: Domains (PDZ (GI: 404931, https://www.ncbi.nlm.nih.gov/protein/404931 ), Cib81 , GSAGx2 , and GSAGx3 ) for insertions were ordered as gBlocks (IDT DNA), and PCR amplified to add on BsaI and linkers (Ala-Ser and Ser-Ala-Gly using primers 77–84 (Supplementary Table ), preceding and following the domain insertion), sites complementary to MuA-BsaI-transposon sites for Golden Gate cloning. .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water.

    Polyacrylamide Gel Electrophoresis:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: FPR1 cDNA was amplified from a cDNA plasmid template (1 μl of glycerol bacterial stock) using a PAGE purified forward primer 2A_FPR1_fwd (GCT ACT AAC TTC TCT CTA TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCA GGT CCT ATG GAG ACA AAT TCC TCT CTC CC) and reverse primer FPR1_rev (GCG GAG GCC ACG CGT CTA CTT TGC CTG TAA CTC CAC C). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Activated Clotting Time Assay:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: FPR1 cDNA was amplified from a cDNA plasmid template (1 μl of glycerol bacterial stock) using a PAGE purified forward primer 2A_FPR1_fwd (GCT ACT AAC TTC TCT CTA TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCA GGT CCT ATG GAG ACA AAT TCC TCT CTC CC) and reverse primer FPR1_rev (GCG GAG GCC ACG CGT CTA CTT TGC CTG TAA CTC CAC C). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For that, RT-PCR product of a defined round was amplified with CFX_HR_fwd (5′-CAA GCT ATA CCA AGC ATA CAA TCA ACT CCA AGC TAG ATC TAC CGG TGG GAG ACG CAA CTG AAT GAA-3′) and CFX_HR_rev (5′-CAA GAA TTG GGA CAA CTC CAG TGA AAA GTT CTT CTC CTT TGC TAG CGT GAC GCG ACT AGT TAC GGA-3′) to attach 46 bp overhang for recombination into pWHE601*. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Plasmid Preparation:

    Article Title: Expression and clinical significance of basic transcription factor 3 in nasopharyngeal carcinoma
    Article Snippet: .. siRNA viral vector construction The BTF3 siRNA transcript template (sense, 5′-GCCGAAGAAGCCTGGGAATCA-3′) was used to generate the viral vector, digested with Age I/Eco RI enzymes (cat. no. R3552L/R3101L; New England BioLabs, Inc., Ipswich, MA, USA), and then ligated with pGCSIL-GFP vector (New England Biolabs). ..

    Article Title: Targeted insertional mutagenesis libraries for deep domain insertion profiling
    Article Snippet: .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research) and eluted with 10 μl water. .. All 10 μl were transformed into 30 μl E. cloni® 10G ELITE electrocompetent cells (Lucigen) in 1.0 mm Biorad cuvettes using a Bio-Rad Gene Pulser II electroporator (settings: 10 μF, 600 Ω, 1.8 kV).

    Article Title: Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
    Article Snippet: .. The resulting PCR products were digested with AgeI-HF (NEB, Ipswich, MA, USA) and ligated into the pRed plasmid to be located between a SV40 promoter and the RFP gene. .. The viral recombinase expression constructs used in the cytotoxicity studies were based on pBabe-puro (Addgene plasmid 1764) and modified to contain an internal ribosomal entry site and a GFP reporter gene as previously described .

    Article Title: Expression and clinical significance of basic transcription factor 3 in nasopharyngeal carcinoma
    Article Snippet: .. The BTF3 siRNA transcript template (sense, 5′-GCCGAAGAAGCCTGGGAATCA-3′) was used to generate the viral vector, digested with Age I/ Eco RI enzymes (cat. no. R3552L/R3101L; New England BioLabs, Inc., Ipswich, MA, USA), and then ligated with pGCSIL-GFP vector (New England Biolabs). ..

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel. .. 100 ng of plasmid backbone was mixed with the PCR-generated tRFP and FPR1 cDNA fragments in equimolar amounts and ligated using 2X Gibson mix (E2611S, NEB).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: .. The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To prepare inserts (candidate SNP-containing sequences), we performed PCR to amplify DNA fragments covering all SNPs selected from SNPs-seq.

    Article Title: Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling
    Article Snippet: .. Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs (Ipswich, MA, USA). pLKO.1‐TRC cloning vector was a gift from David Root (Addgene plasmid # 10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10878). .. Cell Line and Cell Culture Human GCTB Hs 737.T (ATCC, USA) were grown in Dulbecco's modified eagle medium plus 10% FBS, l00 μg/mL streptomycin and penicillin.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. Transformed cells were spread on several SCD-ura plates in a way that assures a moderate colony density which simplifies picking clones.

    Article Title: Domain insertion permissibility-guided engineering of allostery in ion channels
    Article Snippet: .. The product was further digested with AgeI-HF (NEB) and Plasmid-Safe ATP-dependent DNase (Epicentre) to remove any undigested transposon, then cleaned up (Zymo Research), and eluted with 10 µl of water. .. All 10 µl were transformed into 30 µl of Lucigen electrocompetent 10G ELITE E. coli and electroporated in 1.0-mm Biorad cuvettes using a Bio-Rad Gene Pulser II electroporator (settings: 10 µF, 600 Ω, 1.8 kV).

    Agarose Gel Electrophoresis:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel. .. 100 ng of plasmid backbone was mixed with the PCR-generated tRFP and FPR1 cDNA fragments in equimolar amounts and ligated using 2X Gibson mix (E2611S, NEB).

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: .. The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To prepare inserts (candidate SNP-containing sequences), we performed PCR to amplify DNA fragments covering all SNPs selected from SNPs-seq.

    In Vitro:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: Paragraph title: In vitro ribosome repair assay ... For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    CCK-8 Assay:

    Article Title: Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling
    Article Snippet: The CCK‐8 kit was provided by Dojindo Molecular Technologies (Kumamoto, Japan). .. Restriction enzymes EcoRI‐HF and AgeI‐HF were provided by New England Biolabs (Ipswich, MA, USA). pLKO.1‐TRC cloning vector was a gift from David Root (Addgene plasmid # 10878; http://n2t.net/addgene:10878 ; RRID: Addgene_10878).

    CTG Assay:

    Article Title: The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis
    Article Snippet: FPR1 cDNA was amplified from a cDNA plasmid template (1 μl of glycerol bacterial stock) using a PAGE purified forward primer 2A_FPR1_fwd (GCT ACT AAC TTC TCT CTA TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCA GGT CCT ATG GAG ACA AAT TCC TCT CTC CC) and reverse primer FPR1_rev (GCG GAG GCC ACG CGT CTA CTT TGC CTG TAA CTC CAC C). .. PTripz_control plasmid was cleaved using restriction enzymes AgeI_HF (R3552S, Ipswich, MA, USA) and MluI (R0198S) purchased from New England Biolabs (NEB), and gel purified using a 1 % TAE agarose gel.

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For that, RT-PCR product of a defined round was amplified with CFX_HR_fwd (5′-CAA GCT ATA CCA AGC ATA CAA TCA ACT CCA AGC TAG ATC TAC CGG TGG GAG ACG CAA CTG AAT GAA-3′) and CFX_HR_rev (5′-CAA GAA TTG GGA CAA CTC CAG TGA AAA GTT CTT CTC CTT TGC TAG CGT GAC GCG ACT AGT TAC GGA-3′) to attach 46 bp overhang for recombination into pWHE601*. .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

    Gel Extraction:

    Article Title: High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing
    Article Snippet: .. The plasmid vector was linearized by AgeI-HF and SalI-HF digestion (New England Biolabs, Ipswich, MA, USA), followed by agarose gel purification (QIAquick Gel extraction, Qiagen, Germantown, MD, USA) and then 1.0 × AMPure XP DNA bead purification. .. To prepare inserts (candidate SNP-containing sequences), we performed PCR to amplify DNA fragments covering all SNPs selected from SNPs-seq.

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    New England Biolabs agei hf
    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 <t>AgeI</t> used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) <t>RT-PCR</t> performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.
    Agei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Article Snippet: For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    Techniques: Purification, In Vitro, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Ligation, Sequencing