bsaxi  (New England Biolabs)


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    Name:
    BsaXI
    Description:
    BsaXI 500 units
    Catalog Number:
    R0609L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    500 units
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    Structured Review

    New England Biolabs bsaxi
    BsaXI
    BsaXI 500 units
    https://www.bioz.com/result/bsaxi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaxi - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms"

    Article Title: Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

    Journal: bioRxiv

    doi: 10.1101/855452

    Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.
    Figure Legend Snippet: Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing, Expressing, FACS, Marker

    2) Product Images from "Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms"

    Article Title: Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

    Journal: bioRxiv

    doi: 10.1101/855452

    Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.
    Figure Legend Snippet: Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing, Expressing, FACS, Marker

    3) Product Images from "Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes"

    Article Title: Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

    Journal: Clinics

    doi: 10.1590/S1807-59322011000500014

    JAK2 V617F genotyping. (A) PCR amplification of JAK2 : lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles.
    Figure Legend Snippet: JAK2 V617F genotyping. (A) PCR amplification of JAK2 : lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    Related Articles

    Mutagenesis:

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells
    Article Snippet: The plasmid AM1 was constructed by replacing the Renilla luciferase gene of pGL4.75 (Promega, Madison, WI) with the Chroma-Luc™ gene of pCBG68-control plasmid (Promega) at Hin dIII and Xba I sites. .. The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis. .. In the descriptions to follow, the underlined residues illustrate the altered codon base pairs in the luciferase gene.

    Plasmid Preparation:

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells
    Article Snippet: The plasmid AM1 was constructed by replacing the Renilla luciferase gene of pGL4.75 (Promega, Madison, WI) with the Chroma-Luc™ gene of pCBG68-control plasmid (Promega) at Hin dIII and Xba I sites. .. The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis. .. In the descriptions to follow, the underlined residues illustrate the altered codon base pairs in the luciferase gene.

    Purification:

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells
    Article Snippet: The plasmid AM1 was constructed by replacing the Renilla luciferase gene of pGL4.75 (Promega, Madison, WI) with the Chroma-Luc™ gene of pCBG68-control plasmid (Promega) at Hin dIII and Xba I sites. .. The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis. .. In the descriptions to follow, the underlined residues illustrate the altered codon base pairs in the luciferase gene.

    Gel Extraction:

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells
    Article Snippet: The plasmid AM1 was constructed by replacing the Renilla luciferase gene of pGL4.75 (Promega, Madison, WI) with the Chroma-Luc™ gene of pCBG68-control plasmid (Promega) at Hin dIII and Xba I sites. .. The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis. .. In the descriptions to follow, the underlined residues illustrate the altered codon base pairs in the luciferase gene.

    Agarose Gel Electrophoresis:

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells
    Article Snippet: The plasmid AM1 was constructed by replacing the Renilla luciferase gene of pGL4.75 (Promega, Madison, WI) with the Chroma-Luc™ gene of pCBG68-control plasmid (Promega) at Hin dIII and Xba I sites. .. The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis. .. In the descriptions to follow, the underlined residues illustrate the altered codon base pairs in the luciferase gene.

    Article Title: Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes
    Article Snippet: PCR was performed in a 50-µL reaction volume consisting of 100 ng of genomic DNA, 5 µL of 10X reaction buffer, 2 µL of 50 mM MgCl2 , 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers ( ). .. For RFLP analysis, JAK2 and FLT3 PCR products were digested with BsaXI or Eco321 (New England Biolabs, Hitchin, UK), respectively, according to the manufacturer's protocol, and visualized on a 2.5% agarose gel. .. The normal genotype for JAK2 was represented by a 460-bp fragment, and the heterozygous genotype was represented by 460-bp, 241-bp and 189-bp fragments, whereas the homozygous mutant genotype produced 241-bp and 189-bp fragments.

    Article Title: Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms
    Article Snippet: 1-2μl of extracted genomic DNA in a total PCR reaction volume of 25μl were amplified in a 35 cycle PCR using a locus-specific primer set (primer set 1) and AmpliTaq Gold 360 Master Mix (Applied Biosystems) . .. The PCR amplicons were subjected to restriction digestion using BsaXI (New England Biolabs) for 60 minutes at 37°C, and the digested products were run on a 1.5% agarose gel to visualize either the appearance (for WT and F617V clones) or disappearance (for V617F clones) of the digested products. .. The edited locus was PCR-amplified and TA-cloned using the TOPO-TA cloning kit (Invitrogen), and a minimum of 10 white colonies derived through ampicillin-Xgal selection were submitted for sequencing.

    Polymerase Chain Reaction:

    Article Title: Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation
    Article Snippet: The PCR conditions were as follows: an initial denaturation step at 94°C for 3 min; 25 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s; and a final extension step at 72°C for 2 min. .. The PCR products (1 μL) were then digested in a reaction mixture (10 μL) with 0.4 units of Bsa XI (New England BioLabs) at 37°C for 2 h. The digested PCR products were subjected to another round of PCR using the FI and RI-A primers instead of the FO and RO primers. .. The second round of PCR products was digested with Bsa XI, and then 1 μL of the second-digestion samples were subjected to a final PCR to selectively amplify the JAK2 V617F allele in a reaction mixture (15 μL) containing 0.5× Titanium Taq DNA polymerase (TaKaRa Bio), 1× Titanium buffer, 0.2 mM of each dNTP, 1.0 μM FI, 0.1 μM RI-B, 0.5 μM BNA probe, and 0.05 μM Q-Probe.

    Article Title: Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes
    Article Snippet: PCR was performed in a 50-µL reaction volume consisting of 100 ng of genomic DNA, 5 µL of 10X reaction buffer, 2 µL of 50 mM MgCl2 , 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers ( ). .. For RFLP analysis, JAK2 and FLT3 PCR products were digested with BsaXI or Eco321 (New England Biolabs, Hitchin, UK), respectively, according to the manufacturer's protocol, and visualized on a 2.5% agarose gel. .. The normal genotype for JAK2 was represented by a 460-bp fragment, and the heterozygous genotype was represented by 460-bp, 241-bp and 189-bp fragments, whereas the homozygous mutant genotype produced 241-bp and 189-bp fragments.

    Article Title: Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms
    Article Snippet: 1-2μl of extracted genomic DNA in a total PCR reaction volume of 25μl were amplified in a 35 cycle PCR using a locus-specific primer set (primer set 1) and AmpliTaq Gold 360 Master Mix (Applied Biosystems) . .. The PCR amplicons were subjected to restriction digestion using BsaXI (New England Biolabs) for 60 minutes at 37°C, and the digested products were run on a 1.5% agarose gel to visualize either the appearance (for WT and F617V clones) or disappearance (for V617F clones) of the digested products. .. The edited locus was PCR-amplified and TA-cloned using the TOPO-TA cloning kit (Invitrogen), and a minimum of 10 white colonies derived through ampicillin-Xgal selection were submitted for sequencing.

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    New England Biolabs bsaxi
    Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by <t>PCR</t> and <t>BsaXI</t> restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.
    Bsaxi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsaxi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaxi - by Bioz Stars, 2021-05
    95/100 stars
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    Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.

    Journal: bioRxiv

    Article Title: Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

    doi: 10.1101/855452

    Figure Lengend Snippet: Characterization of JAK2 V617F and ‘corrected’ F617V HUDEP clones. (a) DNA gel showing screening of V617F clones by PCR and BsaXI restriction digest. Lower band corresponds to WT allele and higher undigested fragment corresponds to V617F allele. (b) Sanger sequencing traces of V617F homozygous (F1) and heterozygous (H1) clones. (c) Viability curve depicting cell death in the absence of EPO. F1 and F3 V617F homozygote clones exhibited mildly higher cell viability than WT cells. Data is from n=5 independent biological replicates. Mean of all experiments ± SD shown. (d) . Sanger sequencing traces of F617V homozygous (cF1) and heterozygous (cH2) clones. (e) Immunoblot and signal intensity quantification show elevated phosphorylated STAT5 (P-STAT5) and uniform JAK2 expression in JAK2 V617F HUDEP-2 clones without erythropoietin (EPO). Signals were normalized to STAT5 and GAPDH. White bars, JAK2 WT clones; light pink bar, JAK2 V617F heterozygous clone; magenta bars, JAK2 V617F homozygous clones. (f) Immunoblot and signal intensity quantification show elevated phosphorylated STAT1 (P-STAT1) expression in JAK2 V617F HUDEP-2 clones both with and without erythropoietin (EPO). Signals were normalized to STAT1 and GAPDH. White bars, JAK2 WT clones; light pink bars, JAK2 V617F heterozygous clones; magenta bars, JAK2 V617F homozygous clones. (g) Representative FACS plots for gating live HUDEP-2s expressing Kusabira Orange, a marker gene indicative of viable HUDEP-2s. (h) Levels of Glycophorin A (GlyA), an erythroid-specific cell surface marker, in undifferentiated or fully differentiated HUDEP-2s at day 0 and day 5, respectively.

    Article Snippet: The PCR amplicons were subjected to restriction digestion using BsaXI (New England Biolabs) for 60 minutes at 37°C, and the digested products were run on a 1.5% agarose gel to visualize either the appearance (for WT and F617V clones) or disappearance (for V617F clones) of the digested products.

    Techniques: Clone Assay, Polymerase Chain Reaction, Sequencing, Expressing, FACS, Marker

    (A) Transcriptional base substitution in the luciferase gene for the study of SN-BER. The codon 27 of the luciferase gene was modified to a stop codon by introduction of a uracil residue, as indicated. The uracil will be converted to a leucine codon after DNA repair, resulting in luciferase protein expression. (B) The construction of plasmid DNA containing uracil is described under “Materials and methods.” The Renilla luciferase gene of pGL4.75 was replaced by the Chroma-Luc™ gene, and the oligonucleotide fragment containing uracil was ligated at the Bsa XI site introduced by site-directed mutagenesis in the Chroma-Luc™ gene. (C) Confirmation of plasmid preparations. Plasmids AM1, AM1-P (positive) and AM1-U (uracil) were treated with Bsa XI at 37°C for 1 h, and then the mixtures were analyzed by 1% agarose gel electrophoresis. AM1 was used as reference for closed circular (lane 1) and linear DNA (lane 2).

    Journal: DNA repair

    Article Title: DNA polymerase ? and PARP activities in base excision repair in living cells

    doi: 10.1016/j.dnarep.2009.08.004

    Figure Lengend Snippet: (A) Transcriptional base substitution in the luciferase gene for the study of SN-BER. The codon 27 of the luciferase gene was modified to a stop codon by introduction of a uracil residue, as indicated. The uracil will be converted to a leucine codon after DNA repair, resulting in luciferase protein expression. (B) The construction of plasmid DNA containing uracil is described under “Materials and methods.” The Renilla luciferase gene of pGL4.75 was replaced by the Chroma-Luc™ gene, and the oligonucleotide fragment containing uracil was ligated at the Bsa XI site introduced by site-directed mutagenesis in the Chroma-Luc™ gene. (C) Confirmation of plasmid preparations. Plasmids AM1, AM1-P (positive) and AM1-U (uracil) were treated with Bsa XI at 37°C for 1 h, and then the mixtures were analyzed by 1% agarose gel electrophoresis. AM1 was used as reference for closed circular (lane 1) and linear DNA (lane 2).

    Article Snippet: The Bsa XI site was introduced in the Chroma-Luc™ gene by site-directed mutagenesis, and the resultant plasmid was digested by Bsa XI (New England Biolabs, Ipswich, MA) and purified with a gel extraction kit (Qiagen, Valencia, CA) after 1% agarose gel electrophoresis.

    Techniques: Luciferase, Modification, Expressing, Plasmid Preparation, Mutagenesis, Agarose Gel Electrophoresis

    JAK2 V617F genotyping. (A) PCR amplification of JAK2 : lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles.

    Journal: Clinics

    Article Title: Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

    doi: 10.1590/S1807-59322011000500014

    Figure Lengend Snippet: JAK2 V617F genotyping. (A) PCR amplification of JAK2 : lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles.

    Article Snippet: For RFLP analysis, JAK2 and FLT3 PCR products were digested with BsaXI or Eco321 (New England Biolabs, Hitchin, UK), respectively, according to the manufacturer's protocol, and visualized on a 2.5% agarose gel.

    Techniques: Polymerase Chain Reaction, Amplification, Negative Control