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  • 99
    New England Biolabs monarch gel extraction kit
    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic <t>DNA.</t> Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) <t>PCR</t> evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Monarch Gel Extraction Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 100
    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic <t>DNA.</t> Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) <t>PCR</t> evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FLIR Systems flir t1020
    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic <t>DNA.</t> Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) <t>PCR</t> evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Flir T1020, supplied by FLIR Systems, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Journal: Frontiers in Microbiology

    Article Title: Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior

    doi: 10.3389/fmicb.2018.01569

    Figure Lengend Snippet: CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Article Snippet: All PCR products and plasmids were purified using Monarch DNA Gel Extraction Kit (NEB, United States) and MiniBEST Plasmid Purification Kit (Takara, Japan), respectively.

    Techniques: CRISPR, Introduce, Sequencing, Polymerase Chain Reaction, Amplification

    CAST promoter activity and calpastatin isoform expression in MG cells. A , RLM RACE was carried out using 5 μg of total RNA isolated from NFI-hyperphosphorylated (T98) or NFI-hypophosphorylated (U251) MG cells. Antisense primer targeting exon 14 of the CAST gene was used for reverse transcription. Nested PCR amplification was carried out with 5′-ligated outer and inner primers and antisense primers targeting exons 8 and 4 of the CAST gene, respectively. PCR products were electrophoresed in 3% Metaphor (FMC Bioproducts) agarose gel, visualized with ethidium bromide, excised, purified, and sequenced using the BigDye Terminator version 3.1 cycle sequencing kit (Thermo Fisher Scientific). Exon composition of the isolated DNA products based on sequence analysis is indicated on the right. B , domain composition of XL-containing (full-length/type II) and XL-less (type III) calpastatin isoforms. T98 ( C ) and U251 ( D ) MG cells were transiently transfected with luciferase reporter constructs containing the CP (∼2000 bp upstream of CAST exon 1), ALT (in intron 3 containing the NFI-binding sites; ∼4000 bp upstream of CAST exon 4), or empty vector (CNT). Luciferase activity was measured using the Luciferase Assay System (Promega) and the FLUOstar microplate reader (BMG LABTECH). Relative -fold change was calculated relative to the empty vector control. Scatter plots show data from three independent experiments. E , U251 MG cells were transiently transfected with luciferase reporter constructs containing the WT ALT or constructs containing mutation at C2 (ALT-C2*), C3 (ALt-C3*), or both C2 and C3 (ALT-C2*C3*) NFI-binding sites. Luciferase activity was measured 48 h post-transfection as described above. p values were obtained from one-way analysis of variance statistical analysis of three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Effects of nuclear factor I phosphorylation on calpastatin (CAST) gene variant expression and subcellular distribution in malignant glioma cells

    doi: 10.1074/jbc.RA118.004787

    Figure Lengend Snippet: CAST promoter activity and calpastatin isoform expression in MG cells. A , RLM RACE was carried out using 5 μg of total RNA isolated from NFI-hyperphosphorylated (T98) or NFI-hypophosphorylated (U251) MG cells. Antisense primer targeting exon 14 of the CAST gene was used for reverse transcription. Nested PCR amplification was carried out with 5′-ligated outer and inner primers and antisense primers targeting exons 8 and 4 of the CAST gene, respectively. PCR products were electrophoresed in 3% Metaphor (FMC Bioproducts) agarose gel, visualized with ethidium bromide, excised, purified, and sequenced using the BigDye Terminator version 3.1 cycle sequencing kit (Thermo Fisher Scientific). Exon composition of the isolated DNA products based on sequence analysis is indicated on the right. B , domain composition of XL-containing (full-length/type II) and XL-less (type III) calpastatin isoforms. T98 ( C ) and U251 ( D ) MG cells were transiently transfected with luciferase reporter constructs containing the CP (∼2000 bp upstream of CAST exon 1), ALT (in intron 3 containing the NFI-binding sites; ∼4000 bp upstream of CAST exon 4), or empty vector (CNT). Luciferase activity was measured using the Luciferase Assay System (Promega) and the FLUOstar microplate reader (BMG LABTECH). Relative -fold change was calculated relative to the empty vector control. Scatter plots show data from three independent experiments. E , U251 MG cells were transiently transfected with luciferase reporter constructs containing the WT ALT or constructs containing mutation at C2 (ALT-C2*), C3 (ALt-C3*), or both C2 and C3 (ALT-C2*C3*) NFI-binding sites. Luciferase activity was measured 48 h post-transfection as described above. p values were obtained from one-way analysis of variance statistical analysis of three independent experiments. *, p

    Article Snippet: The DNA was electrophoresed in a 3% MetaPhor agarose (FMC Bioproducts, Rockland, ME) gel, excised, purified (Monarch DNA extraction kit, New England Biolabs), and sequenced (BigDye Terminator® version 3.1 cycle sequencing kit, Thermo Fisher Scientific).

    Techniques: Activity Assay, Expressing, Isolation, Nested PCR, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, Sequencing, Transfection, Luciferase, Construct, Binding Assay, Plasmid Preparation, Mutagenesis

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Journal: Nature Communications

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    doi: 10.1038/s41467-018-07611-1

    Figure Lengend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Article Snippet: ThermoPol buffer, Taq DNA polymerase, G. stearothermophilus Bst DNA polymerase, LF, and its variants Bst 2.0 DNA polymerase (2.0), and Bst 3.0 DNA polymerase (3.0), DH5α competent cells, and Monarch DNA gel extraction kits were purchased from New England Biolabs (Ipswich, MA).

    Techniques: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis