aari  (Thermo Fisher)


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  • 99
    Name:
    AarI
    Description:
    5'  C  A  C  C  T  G  C  N4↓ 3'3'  G  T  G  G  A  C  G  N8↑ 5'Thermo Scientific AarI restriction enzyme recognizes CACCTGC(4/8)^ sites and cuts best at 37°C in its own unique (+oligo) buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.Features• Superior quality—stringent quality control and industry leading manufacturing process• Convenient color-coded Five Buffer System• Includes universal Tango buffer for double-digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism (RFLP)• SNPNote: For methylation sensitivity, refer to product specifications. For cleavage with AarI at least two copies of its recognition sequence are required. Inclusion of 0.5 µM oligonucleotide with the AarI recognition sequence in the reaction mixture significantly improves cleavage of DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve. Greater than 10-fold overdigestion with AarI may result in star activity. AarI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
    Catalog Number:
    ER1581
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Size:
    25 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, Restriction Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher aari
    5'  C  A  C  C  T  G  C  N4↓ 3'3'  G  T  G  G  A  C  G  N8↑ 5'Thermo Scientific AarI restriction enzyme recognizes CACCTGC(4/8)^ sites and cuts best at 37°C in its own unique (+oligo) buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.Features• Superior quality—stringent quality control and industry leading manufacturing process• Convenient color-coded Five Buffer System• Includes universal Tango buffer for double-digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism (RFLP)• SNPNote: For methylation sensitivity, refer to product specifications. For cleavage with AarI at least two copies of its recognition sequence are required. Inclusion of 0.5 µM oligonucleotide with the AarI recognition sequence in the reaction mixture significantly improves cleavage of DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve. Greater than 10-fold overdigestion with AarI may result in star activity. AarI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
    https://www.bioz.com/result/aari/product/Thermo Fisher
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    aari - by Bioz Stars, 2019-10
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Paragraph title: CRISPRa and orthogonal sgRNA library cloning ... The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: Paragraph title: sgRNA library cloning ... The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: Paragraph title: sgRNA Library Design and Cloning. ... The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Paragraph title: 4.2. Cloning and Construction of Yeast Plasmids ... Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Article Title: Recombinant expression of twelve evolutionarily diverse subfamily I? aminotransferases
    Article Snippet: We report here the purification and initial kinetic investigations of eleven additional members of this group. .. All cloning enzymes were obtained from New England Biolabs (Ipswich, MA) except that alkaline phosphatase was from USB (Cleveland, OH), Pfu polymerase was from Stratagene (La Jolla, CA), AarI was from Fermentas (Burlington, Ontario, Canada), and all GATEWAY cloning reagents, including enzymes, were from Invitrogen (Carlsbad, CA). .. Purification of DNA fragments was carried out using GFX kits from GE Healthcare (Piscataway, NJ).

    Amplification:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Template pools were PCR amplified using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific) according to the manufacturers protocol with 1 ng/uL sgRNA template DNA, 1 uM forward primer (5′-GGAGAACCACCTTGTTGG-3′), 1 uM reverse primer (5′-GTTTCCAGCATAGCTCTTAAAC-3′) and the following cycle numbers: 1x (98C for 3 min), 15x (98C for 1 sec, 55C for 15 sec, 72C for 20 sec) and 1x (72C for 5 min). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: Template pools were PCR amplified using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific) according to the manufacturers protocol with 1 ng/μL sgRNA template DNA, 1 μM forward primer (5’-GGAGAACCACCTTGTTGG-3′), 1 μM reverse primer (5′- GTTTCCAGCATAGCTCTTAAAC-3′) and the following cycle numbers: 1x (98C for 3 min), 15x (98C for 1 s, 55C for 15 s, 72C for 20 s) and 1x (72C for 5 min). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Coding sequences from T. wilfordii encoding native diterpene synthases (diTPS) TwTPS9 and TwTPS27 were PCR amplified. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Synthesized:

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: The synthesized sgRNA template sequences were of the format: 5′-GGAGAACCACCTTGTTGG-(N)20 -GTTTAAGAGCTATGCTGGAAAC-3′. .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Construct:

    Article Title: CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis
    Article Snippet: For CRISPR-Cas9 mediated editing efficiency analysis, plasmids targeting the α-amylase amyE in B . subtilis were constructed. .. Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides.

    Electrophoresis:

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Electrophoresis of PCR products was carried out on 1% (w /v ) agarose gels and corresponding bands were cut out and purified using a Zymoclean™ Gel DNA recovery kit (Zymo Research) following standard protocol. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Microarray:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: For the CRISPRa library, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based sgLenti sgRNA library vector (see for vector map). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: For both the CRISPRwt and CRISPRi libraries, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based barcoded sgLenti sgRNA library vector (see Additional file for vector map). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: The 20-nt target-specific sgRNA sequences were synthesized as a pool on microarray surfaces (CustomArray), flanked by overhangs compatible with Gibson Assembly into the pSico-based barcoded sgLenti sgRNA library vector (Addgene #105996). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Incubation:

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template.

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD). .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD).

    Luciferase:

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: RNAs after hDcp2 treatment were resolved electrophoretically on denaturing 15% acrylamide / 7 M urea 1× TBE gel and were stained with SYBR Gold (Invitrogen) and visualized using a Typhoon FLA 9500 (GE Healthcare). .. mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. This plasmid was obtained by cloning sequence encoding firefly luciferase, two repeats of β-globin 3′UTR and 128 adenines from hRLuc-pRNA2(A)128 plasmid ( ) into pJET1.2 vector (ThermoFisher Scientific).

    Activity Assay:

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis
    Article Snippet: BsaI, BbsI, BsmBI and BtgZI were purchased from NEB, AarI from Thermo ScientificTM . .. BsaI, BbsI, BsmBI and BtgZI were purchased from NEB, AarI from Thermo ScientificTM .

    In Silico:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: We introduced between 2 and 7 synonymous mutations simultaneously in silico to the identified regions and reassessed structural stability or pseudoknot formation using the same structure prediction programs. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Expressing:

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Dual expression vectors derived from the pSP-G series [ ] were mainly used for gene assembly ( ) except single expression vector pEVE3. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Modification:

    Article Title: CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis
    Article Snippet: Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides. .. Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides.

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: The third PCR also used the Starter Primers and Pfu Turbo polymerase under the thermocycling conditions of the first PCR. .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD). .. Ligations were performed with T4 DNA Ligase (Invitrogen, Carlsbad, CA) overnight at 14°C.

    Transformation Assay:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Derivative Assay:

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Dual expression vectors derived from the pSP-G series [ ] were mainly used for gene assembly ( ) except single expression vector pEVE3. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Electroporation:

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD). .. The final ligation reaction was diluted 1:5 in distilled water and 5 μl was used to transform 20 μl of ElectroMax DH10B E . coli cells (Invitrogen).

    Inverse PCR:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Mutations were introduced into pHW2000 bidirectional plasmids by inverse PCR with primers (see Supplementary Table ) including selected mutations and unique ligation sites. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Ligation:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Mutations were introduced into pHW2000 bidirectional plasmids by inverse PCR with primers (see Supplementary Table ) including selected mutations and unique ligation sites. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: Following ligation with T4 DNA ligase, Opener Primer pairs ( ) and Pfu Turbo polymerase (Stratagene) were used to generate the three Intermediate DNAs under these conditions: denaturation at 95° for 2 min followed by 25 cycles of 95°C, 20 sec; 51°C, 30 sec; and 72°C, 3 min, followed by 72°C, 10 min, and finally 4°C. .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD).

    Buffer Exchange:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 uL reaction volume.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 μL reaction volume.

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using a Gibson Assmbly Master Mix (New England Biolabs), 1,000-ng digested sgLenti and 100-ng amplified sgRNA library insert were assembled in a total 200-µL reaction volume.

    Cell Culture:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The reaction was purified using P-30 buffer exchange columns (Biorad) that were equilibrated 5x with H2 O and the total eluted volume was transformed into three vials of Electromax DH5α (ThermoFisher).

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: Paragraph title: Cell culture and in vivo measurements of translation efficiency ... mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The reaction was purified using P-30 buffer exchange columns (Biorad) that were equilibrated 5x with H2 O and the total eluted volume was transformed into three vials of Electromax DH5α (ThermoFisher).

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. The reaction was purified using P-30 buffer exchange columns (Bio-Rad) that were equilibrated 5× with H2 O and the total eluted volume was transformed into three vials of Electromax DH5α (ThermoFisher).

    Generated:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Once predicted destabilizing mutations were identified, mutant viruses were generated. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    DNA Sequencing:

    Article Title: Recombinant expression of twelve evolutionarily diverse subfamily I? aminotransferases
    Article Snippet: All cloning enzymes were obtained from New England Biolabs (Ipswich, MA) except that alkaline phosphatase was from USB (Cleveland, OH), Pfu polymerase was from Stratagene (La Jolla, CA), AarI was from Fermentas (Burlington, Ontario, Canada), and all GATEWAY cloning reagents, including enzymes, were from Invitrogen (Carlsbad, CA). .. DNA plasmid purification was done with Wizard Miniprep or Midiprep kits from Promega (Madison, WI).

    Polymerase Chain Reaction:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Mutations were introduced into pHW2000 bidirectional plasmids by inverse PCR with primers (see Supplementary Table ) including selected mutations and unique ligation sites. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid. .. Digested PCR products were PCR purified and ligated using Instant Sticky End Ligase (NEB).

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: The third PCR also used the Starter Primers and Pfu Turbo polymerase under the thermocycling conditions of the first PCR. .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD).

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Coding sequences from T. wilfordii encoding native diterpene synthases (diTPS) TwTPS9 and TwTPS27 were PCR amplified. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    In Vivo:

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: Paragraph title: Cell culture and in vivo measurements of translation efficiency ... mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template.

    RNA Sequencing Assay:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: We also identified four regions in segment 5, amenable to extensive silent mutagenesis that were either highly bound or represented at the same frequency in PAR-CLIP and RNA-seq data sets. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Mutagenesis:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Once predicted destabilizing mutations were identified, mutant viruses were generated. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Size-exclusion Chromatography:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Template pools were PCR amplified using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific) according to the manufacturers protocol with 1 ng/uL sgRNA template DNA, 1 uM forward primer (5′-GGAGAACCACCTTGTTGG-3′), 1 uM reverse primer (5′-GTTTCCAGCATAGCTCTTAAAC-3′) and the following cycle numbers: 1x (98C for 3 min), 15x (98C for 1 sec, 55C for 15 sec, 72C for 20 sec) and 1x (72C for 5 min). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel).

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: Following ligation with T4 DNA ligase, Opener Primer pairs ( ) and Pfu Turbo polymerase (Stratagene) were used to generate the three Intermediate DNAs under these conditions: denaturation at 95° for 2 min followed by 25 cycles of 95°C, 20 sec; 51°C, 30 sec; and 72°C, 3 min, followed by 72°C, 10 min, and finally 4°C. .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD).

    Purification:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 uL reaction volume.

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. Following 3 h incubation, 1 U/μl DNase I (ThermoFisher Scientific) was added and incubation was continued for 30 min at 37°C, after that EDTA was added to 25 μM final concentration.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 μL reaction volume.

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using a Gibson Assmbly Master Mix (New England Biolabs), 1,000-ng digested sgLenti and 100-ng amplified sgRNA library insert were assembled in a total 200-µL reaction volume.

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Electrophoresis of PCR products was carried out on 1% (w /v ) agarose gels and corresponding bands were cut out and purified using a Zymoclean™ Gel DNA recovery kit (Zymo Research) following standard protocol. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Article Title: Recombinant expression of twelve evolutionarily diverse subfamily I? aminotransferases
    Article Snippet: All cloning enzymes were obtained from New England Biolabs (Ipswich, MA) except that alkaline phosphatase was from USB (Cleveland, OH), Pfu polymerase was from Stratagene (La Jolla, CA), AarI was from Fermentas (Burlington, Ontario, Canada), and all GATEWAY cloning reagents, including enzymes, were from Invitrogen (Carlsbad, CA). .. Purification of DNA fragments was carried out using GFX kits from GE Healthcare (Piscataway, NJ).

    Sequencing:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: All plasmids used contained a CEN/ARS (centromere and autonomously replicating sequence). .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    CRISPR:

    Article Title: CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis
    Article Snippet: For CRISPR-Cas9 mediated editing efficiency analysis, plasmids targeting the α-amylase amyE in B . subtilis were constructed. .. Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides.

    Concentration Assay:

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. Typical in vitro transcription reaction (20 μl) was incubated in 40°C for 3 h and contained: RNA Pol Buffer,1 U/μl SP6 polymerase, 1 U/μl RiboLock RNase Inhibitor, 0.5 mM ATP/CTP/UTP, 0.125 mM GTP, 1.25 mM cap analogue and 0.1 μg of digested plasmid as a template.

    Plasmid Preparation:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 uL reaction volume.

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: RNAs after hDcp2 treatment were resolved electrophoretically on denaturing 15% acrylamide / 7 M urea 1× TBE gel and were stained with SYBR Gold (Invitrogen) and visualized using a Typhoon FLA 9500 (GE Healthcare). .. mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. This plasmid was obtained by cloning sequence encoding firefly luciferase, two repeats of β-globin 3′UTR and 128 adenines from hRLuc-pRNA2(A)128 plasmid ( ) into pJET1.2 vector (ThermoFisher Scientific).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Mutations were introduced into pHW2000 bidirectional plasmids by inverse PCR with primers (see Supplementary Table ) including selected mutations and unique ligation sites. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid. .. Digested PCR products were PCR purified and ligated using Instant Sticky End Ligase (NEB).

    Article Title: CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis
    Article Snippet: For CRISPR-Cas9 mediated editing efficiency analysis, plasmids targeting the α-amylase amyE in B . subtilis were constructed. .. Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides. .. The annealed oligonucleotides consisted of the 20 nucleotide protospacer region for targeting of the Cas9 protein and 4 nucleotides to generate a single stranded overhang for compatibility with the AarI digested vector.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 μL reaction volume.

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning
    Article Snippet: The third PCR also used the Starter Primers and Pfu Turbo polymerase under the thermocycling conditions of the first PCR. .. Restriction digestion of the parent plasmid and the Linear Modified DNAs were done using enzymes XhoI (New England Biolabs) and AarI (Fermentas, Glen Burnie, MD). .. Ligations were performed with T4 DNA Ligase (Invitrogen, Carlsbad, CA) overnight at 14°C.

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using a Gibson Assmbly Master Mix (New England Biolabs), 1,000-ng digested sgLenti and 100-ng amplified sgRNA library insert were assembled in a total 200-µL reaction volume.

    Article Title: Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast
    Article Snippet: Dual expression vectors derived from the pSP-G series [ ] were mainly used for gene assembly ( ) except single expression vector pEVE3. .. Yeast codon optimized versions ( ) of TwTPS9 and TwTPS27 , designed to exclude the endonuclease sites HindIII, SacII, XhoI, SpeI, SacI, AarI, and PmeI were provided by GeneArt® (Thermofisher Scientific, Zug, Switzerland).

    Article Title: Recombinant expression of twelve evolutionarily diverse subfamily I? aminotransferases
    Article Snippet: All cloning enzymes were obtained from New England Biolabs (Ipswich, MA) except that alkaline phosphatase was from USB (Cleveland, OH), Pfu polymerase was from Stratagene (La Jolla, CA), AarI was from Fermentas (Burlington, Ontario, Canada), and all GATEWAY cloning reagents, including enzymes, were from Invitrogen (Carlsbad, CA). .. Purification of DNA fragments was carried out using GFX kits from GE Healthcare (Piscataway, NJ).

    Agarose Gel Electrophoresis:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was preapred by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 uL reaction volume.

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. Following 3 h incubation, 1 U/μl DNase I (ThermoFisher Scientific) was added and incubation was continued for 30 min at 37°C, after that EDTA was added to 25 μM final concentration.

    Article Title: Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using Gibson Assmbly Master Mix (NEB), 1000 ng digested sgLenti and 100 ng amplified sgRNA library insert were assembled in a total 200 μL reaction volume.

    Article Title: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation
    Article Snippet: PCR products were purified using Minelute columns (Qiagen). .. The library vector sgLenti was prepared by restriction digest with AarI (Thermo-Fischer) at 37 °C overnight, followed by 1% agarose gel excision of the digested band and purification via NucleoSpin columns (Macherey-Nagel). .. Using a Gibson Assmbly Master Mix (New England Biolabs), 1,000-ng digested sgLenti and 100-ng amplified sgRNA library insert were assembled in a total 200-µL reaction volume.

    In Vitro:

    Article Title: mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation
    Article Snippet: RNAs after hDcp2 treatment were resolved electrophoretically on denaturing 15% acrylamide / 7 M urea 1× TBE gel and were stained with SYBR Gold (Invitrogen) and visualized using a Typhoon FLA 9500 (GE Healthcare). .. mRNAs encoding firefly luciferase for measurement of translational efficiency in HeLa cells were synthetized by in vitro transcription with SP6 polymerase using pJET_luc_128A plasmid digested with AarI (ThermoFisher Scientifics) as a template. .. This plasmid was obtained by cloning sequence encoding firefly luciferase, two repeats of β-globin 3′UTR and 128 adenines from hRLuc-pRNA2(A)128 plasmid ( ) into pJET1.2 vector (ThermoFisher Scientific).

    Activation Assay:

    Article Title: CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis
    Article Snippet: Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides. .. Plasmid pBAC0015 was digested with AarI (Thermo Fisher Scientific) to remove the kanamycin resistance gene and ligated to two annealed and phosphorylated DNA oligonucleotides of 24 nucleotides.

    Variant Assay:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: We selected variant codon combinations that would disrupt or maintain the predicted vRNA structure but not change the encoded amino acid, alter codon usage, or disrupt alternative reading frames or splicing events. .. PCR products were gel extracted and digested with either BsmbI (New England Biolabs, (NEB)) or AarI (Thermo Fisher Scientific) restriction enzymes and DpnI (NEB) to remove residual parent plasmid.

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    Thermo Fisher aari 2 u µl
    Flowchart for isolation of Cas9-free target mutants. For construction using <t>pKIR1.1,</t> hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).
    Aari 2 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aari 2 u µl/product/Thermo Fisher
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    aari 2 u µl - by Bioz Stars, 2019-10
    91/100 stars
      Buy from Supplier

    99
    Thermo Fisher aari
    Flowchart for isolation of Cas9-free target mutants. For construction using <t>pKIR1.1,</t> hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).
    Aari, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aari/product/Thermo Fisher
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    aari - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Flowchart for isolation of Cas9-free target mutants. For construction using pKIR1.1, hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).

    Journal: Plant and Cell Physiology

    Article Title: pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

    doi: 10.1093/pcp/pcw191

    Figure Lengend Snippet: Flowchart for isolation of Cas9-free target mutants. For construction using pKIR1.1, hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).

    Article Snippet: For pKIR1.1_PSD3, pKIR1.1_ADH1-2 and pKI1.1R_PDS3, pKIR1.1 or pKI1.1R was digested by Aar I (Thermo Fisher Scientific, #ER1581) with alkaline phosphatase rAPid (Roche, #04898133001).

    Techniques: Isolation, Sequencing, Fluorescence, Selection, Mutagenesis

    pKIR1.1 construction. (A) For pKIR1.1, U6.26p::2 × AarI:sgRNA was inserted into the Sbf I site of pKIR1.0. (B) Flowchart of pKIR1.1-based vector construction. The upper sequence is an enlarged view of the boundary (yellow) between the U6.26 promoter (pale green) and the sgRNA scaffold (orange). This boundary contains an inverted repeat of Aar I recognition sites, and Aar I digestion generates four-base overhangs. A hybridized primer with overhangs can be inserted into this site as shown. G with a red triangle is the first base for RNA transcription from the U6.26 promoter. G + (N) 19 indicates the target sequence.

    Journal: Plant and Cell Physiology

    Article Title: pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

    doi: 10.1093/pcp/pcw191

    Figure Lengend Snippet: pKIR1.1 construction. (A) For pKIR1.1, U6.26p::2 × AarI:sgRNA was inserted into the Sbf I site of pKIR1.0. (B) Flowchart of pKIR1.1-based vector construction. The upper sequence is an enlarged view of the boundary (yellow) between the U6.26 promoter (pale green) and the sgRNA scaffold (orange). This boundary contains an inverted repeat of Aar I recognition sites, and Aar I digestion generates four-base overhangs. A hybridized primer with overhangs can be inserted into this site as shown. G with a red triangle is the first base for RNA transcription from the U6.26 promoter. G + (N) 19 indicates the target sequence.

    Article Snippet: For pKIR1.1_PSD3, pKIR1.1_ADH1-2 and pKI1.1R_PDS3, pKIR1.1 or pKI1.1R was digested by Aar I (Thermo Fisher Scientific, #ER1581) with alkaline phosphatase rAPid (Roche, #04898133001).

    Techniques: Plasmid Preparation, Sequencing