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    Name:
    XmaI
    Description:
    XmaI 2 500 units
    Catalog Number:
    r0180l
    Price:
    282
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs xma i
    XmaI
    XmaI 2 500 units
    https://www.bioz.com/result/xma i/product/New England Biolabs
    Average 99 stars, based on 212 article reviews
    Price from $9.99 to $1999.99
    xma i - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4"

    Article Title: Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01670-6

    Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side
    Figure Legend Snippet: Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

    Techniques Used: Expressing, Transgenic Assay, Southern Blot, Molecular Weight, Marker

    2) Product Images from "Disruption of the Rag-Ragulator complex by c17orf59 inhibits mTORC1"

    Article Title: Disruption of the Rag-Ragulator complex by c17orf59 inhibits mTORC1

    Journal: Cell reports

    doi: 10.1016/j.celrep.2015.07.052

    c17orf59 localizes to lysosomes with Ragulator
    Figure Legend Snippet: c17orf59 localizes to lysosomes with Ragulator

    Techniques Used:

    Overexpression of c17orf59 inhibits mTORC1
    Figure Legend Snippet: Overexpression of c17orf59 inhibits mTORC1

    Techniques Used: Over Expression

    c17orf59 is a Ragulator-interacting protein
    Figure Legend Snippet: c17orf59 is a Ragulator-interacting protein

    Techniques Used:

    Loss of c17orf59 does not alter mTORC1 signaling in response to amino acids or insulin
    Figure Legend Snippet: Loss of c17orf59 does not alter mTORC1 signaling in response to amino acids or insulin

    Techniques Used:

    c17orf59 disrupts the Rag-Ragulator interaction
    Figure Legend Snippet: c17orf59 disrupts the Rag-Ragulator interaction

    Techniques Used:

    3) Product Images from "Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis"

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

    Journal: Nature protocols

    doi: 10.1038/nprot.2010.131

    Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.
    Figure Legend Snippet: Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    4) Product Images from "Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat"

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    Journal: The Crispr Journal

    doi: 10.1089/crispr.2017.0010

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Figure Legend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Techniques Used: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    5) Product Images from "Fluorescent labeling of tetracysteine-tagged proteins in intact cells"

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    Journal: Nature protocols

    doi: 10.1038/nprot.2010.129

    Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.
    Figure Legend Snippet: Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.

    Techniques Used: Labeling, Transfection, Staining, Imaging

    Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.
    Figure Legend Snippet: Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.

    Techniques Used: Labeling, Construct, Transfection, Fluorescence, Transmission Assay

    6) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    7) Product Images from "Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat"

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    Journal: The Crispr Journal

    doi: 10.1089/crispr.2017.0010

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Figure Legend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Techniques Used: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    8) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    9) Product Images from "Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus"

    Article Title: Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

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

    doi:

    Strategy for the preparation of a double-stranded DNA fragment coupled to an NLS peptide. A functional luciferase gene of 3,380 bp was cut out of pCMVLuc with Xma I and Sal I. Further digestion with Xmn I and Bsp HI cut the unwanted restriction fragment into small fragments (970, 875, 768, and 240 bp) that were removed by sucrose gradient centrifugation. The capped CMVLuc-NLS DNA was obtained by ligation of the 32 P-labeled (∗) oligonucleotide-peptide and oligonucleotide-cap hairpins to the restriction fragment.
    Figure Legend Snippet: Strategy for the preparation of a double-stranded DNA fragment coupled to an NLS peptide. A functional luciferase gene of 3,380 bp was cut out of pCMVLuc with Xma I and Sal I. Further digestion with Xmn I and Bsp HI cut the unwanted restriction fragment into small fragments (970, 875, 768, and 240 bp) that were removed by sucrose gradient centrifugation. The capped CMVLuc-NLS DNA was obtained by ligation of the 32 P-labeled (∗) oligonucleotide-peptide and oligonucleotide-cap hairpins to the restriction fragment.

    Techniques Used: Functional Assay, Luciferase, Gradient Centrifugation, Ligation, Labeling

    10) Product Images from "Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae"

    Article Title: Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

    Journal: Plasmid

    doi: 10.1016/j.plasmid.2014.09.005

    Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.
    Figure Legend Snippet: Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.

    Techniques Used: Modification, Positron Emission Tomography, Clone Assay, Plasmid Preparation, Amplification, Transformation Assay, Homologous Recombination, In Vivo, Expressing, Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. Screening of gw2 knockout mutants To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine
    Article Snippet: .. The amplified PCR product was digested with XmaI and PacI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBN previously digested with XmaI and PacI . .. The resulting plasmid was designated pSPBN-LBVG ( ).

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Southern Blot:

    Article Title: Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4
    Article Snippet: .. Southern blot analysis For determination of transgene copy number integration by Southern analysis, total nucleic acid was extracted from banana leaf tissue, treated with RNAse A, and 15 μg aliquots of genomic DNA were digested overnight with 20 U of restriction enzyme Hin dIII or Xma I (New England Biolabs) overnight at 37 °C. .. Digested DNA was electrophoresed through 0.9% agarose gels, transferred to a nylon membrane (Roche), and UV cross-linked.

    Labeling:

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells
    Article Snippet: .. Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents. .. EDT (1,2-ethanedithiol, grade for synthesis > 99%; Merck, cat. no. 8.00795.0100) The compound has a bad odor.

    Mouse Assay:

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis
    Article Snippet: .. Both available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) pSV.RecA vector: available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) Swiss Webster female mice (Taconic) Bacterial artificial chromosomes (BACs in DH10B host bacteria; Invitrogen Corporation, BACPAC Resources at CHORI or Riken Bioresources Center) PIR1 chemically competent E. coli (Invitrogen, cat. no. C1010-10) PIR2 chemically competent E. coli (Invitrogen, cat. no. C1111-10) MAX efficiency DH5α-competent cells (Invitrogen, cat. no. 18258-012) PCR primers (Invitrogen and Biosynthesis; see REAGENT SETUP) AscI restriction endonuclease (New England Biolabs, cat. no. R0558L) EcoRI restriction endonuclease (New England Biolabs, cat. no. R0101L) MluI restriction endonuclease (New England Biolabs, cat. no. R0198L) SwaI restriction endonuclease (New England Biolabs, cat. no. R0604L) T4 DNA ligase (New England Biolabs, cat. no. M0202L) XmaI restriction endonuclease (New England Biolabs, cat. no. R0180L) PI-SceI endonuclease (New England Biolabs, cat. no. R0696S) λ-DNA-HindIII Digest (New England Biolabs, cat. no. N3012S) Low-range PFG marker DNA ladder (New England Biolabs, cat. no. NO350S) 2-log DNA ladder (New England Biolabs, cat. no. N3200L) 1-Butanol (Fisher Scientific, cat. no. A399) 2-Propanol (Isopropanol, Fisher Scientific, cat. no. A416) Ammonium acetate (Fisher Scientific, cat. no. A637) Ampicillin (amp; Sigma-Aldrich, cat. no. A9518; see REAGENT SETUP) Calcium chloride dihydrate (CaCl2 , Fisher Scientific, cat. no. C70–500) Cesium chloride (CsCl, Fischer Scientific, cat. no. BP1595-500) Chloramphenicol (chlor; Sigma-Aldrich, cat. no. C0378; see REAGENT SETUP) Chloroform (Fisher Scientific, cat. no. C298–500) Ethidium bromide solution (10 mg ml−1 ; Sigma-Aldrich, cat. no. E1510) ! .. Ethanol (Pharmco-AAPER) EDTA (Sigma-Aldrich, cat. no. E5134) FailSafe PCR System (Epicentre, cat. no. FS99250) Glacial acetic acid (Fisher Scientific, cat. no. A38S) !

    Polymerase Chain Reaction:

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis
    Article Snippet: .. Both available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) pSV.RecA vector: available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) Swiss Webster female mice (Taconic) Bacterial artificial chromosomes (BACs in DH10B host bacteria; Invitrogen Corporation, BACPAC Resources at CHORI or Riken Bioresources Center) PIR1 chemically competent E. coli (Invitrogen, cat. no. C1010-10) PIR2 chemically competent E. coli (Invitrogen, cat. no. C1111-10) MAX efficiency DH5α-competent cells (Invitrogen, cat. no. 18258-012) PCR primers (Invitrogen and Biosynthesis; see REAGENT SETUP) AscI restriction endonuclease (New England Biolabs, cat. no. R0558L) EcoRI restriction endonuclease (New England Biolabs, cat. no. R0101L) MluI restriction endonuclease (New England Biolabs, cat. no. R0198L) SwaI restriction endonuclease (New England Biolabs, cat. no. R0604L) T4 DNA ligase (New England Biolabs, cat. no. M0202L) XmaI restriction endonuclease (New England Biolabs, cat. no. R0180L) PI-SceI endonuclease (New England Biolabs, cat. no. R0696S) λ-DNA-HindIII Digest (New England Biolabs, cat. no. N3012S) Low-range PFG marker DNA ladder (New England Biolabs, cat. no. NO350S) 2-log DNA ladder (New England Biolabs, cat. no. N3200L) 1-Butanol (Fisher Scientific, cat. no. A399) 2-Propanol (Isopropanol, Fisher Scientific, cat. no. A416) Ammonium acetate (Fisher Scientific, cat. no. A637) Ampicillin (amp; Sigma-Aldrich, cat. no. A9518; see REAGENT SETUP) Calcium chloride dihydrate (CaCl2 , Fisher Scientific, cat. no. C70–500) Cesium chloride (CsCl, Fischer Scientific, cat. no. BP1595-500) Chloramphenicol (chlor; Sigma-Aldrich, cat. no. C0378; see REAGENT SETUP) Chloroform (Fisher Scientific, cat. no. C298–500) Ethidium bromide solution (10 mg ml−1 ; Sigma-Aldrich, cat. no. E1510) ! .. Ethanol (Pharmco-AAPER) EDTA (Sigma-Aldrich, cat. no. E5134) FailSafe PCR System (Epicentre, cat. no. FS99250) Glacial acetic acid (Fisher Scientific, cat. no. A38S) !

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. Screening of gw2 knockout mutants To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine
    Article Snippet: .. The amplified PCR product was digested with XmaI and PacI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBN previously digested with XmaI and PacI . .. The resulting plasmid was designated pSPBN-LBVG ( ).

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Knock-Out:

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. Screening of gw2 knockout mutants To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat
    Article Snippet: .. To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB). .. If all three copies of the GW2T2 target site are mutated by CRISPR-Cas9, the PCR products should not be digested.

    Marker:

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis
    Article Snippet: .. Both available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) pSV.RecA vector: available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) Swiss Webster female mice (Taconic) Bacterial artificial chromosomes (BACs in DH10B host bacteria; Invitrogen Corporation, BACPAC Resources at CHORI or Riken Bioresources Center) PIR1 chemically competent E. coli (Invitrogen, cat. no. C1010-10) PIR2 chemically competent E. coli (Invitrogen, cat. no. C1111-10) MAX efficiency DH5α-competent cells (Invitrogen, cat. no. 18258-012) PCR primers (Invitrogen and Biosynthesis; see REAGENT SETUP) AscI restriction endonuclease (New England Biolabs, cat. no. R0558L) EcoRI restriction endonuclease (New England Biolabs, cat. no. R0101L) MluI restriction endonuclease (New England Biolabs, cat. no. R0198L) SwaI restriction endonuclease (New England Biolabs, cat. no. R0604L) T4 DNA ligase (New England Biolabs, cat. no. M0202L) XmaI restriction endonuclease (New England Biolabs, cat. no. R0180L) PI-SceI endonuclease (New England Biolabs, cat. no. R0696S) λ-DNA-HindIII Digest (New England Biolabs, cat. no. N3012S) Low-range PFG marker DNA ladder (New England Biolabs, cat. no. NO350S) 2-log DNA ladder (New England Biolabs, cat. no. N3200L) 1-Butanol (Fisher Scientific, cat. no. A399) 2-Propanol (Isopropanol, Fisher Scientific, cat. no. A416) Ammonium acetate (Fisher Scientific, cat. no. A637) Ampicillin (amp; Sigma-Aldrich, cat. no. A9518; see REAGENT SETUP) Calcium chloride dihydrate (CaCl2 , Fisher Scientific, cat. no. C70–500) Cesium chloride (CsCl, Fischer Scientific, cat. no. BP1595-500) Chloramphenicol (chlor; Sigma-Aldrich, cat. no. C0378; see REAGENT SETUP) Chloroform (Fisher Scientific, cat. no. C298–500) Ethidium bromide solution (10 mg ml−1 ; Sigma-Aldrich, cat. no. E1510) ! .. Ethanol (Pharmco-AAPER) EDTA (Sigma-Aldrich, cat. no. E5134) FailSafe PCR System (Epicentre, cat. no. FS99250) Glacial acetic acid (Fisher Scientific, cat. no. A38S) !

    Plasmid Preparation:

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis
    Article Snippet: .. Both available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) pSV.RecA vector: available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) Swiss Webster female mice (Taconic) Bacterial artificial chromosomes (BACs in DH10B host bacteria; Invitrogen Corporation, BACPAC Resources at CHORI or Riken Bioresources Center) PIR1 chemically competent E. coli (Invitrogen, cat. no. C1010-10) PIR2 chemically competent E. coli (Invitrogen, cat. no. C1111-10) MAX efficiency DH5α-competent cells (Invitrogen, cat. no. 18258-012) PCR primers (Invitrogen and Biosynthesis; see REAGENT SETUP) AscI restriction endonuclease (New England Biolabs, cat. no. R0558L) EcoRI restriction endonuclease (New England Biolabs, cat. no. R0101L) MluI restriction endonuclease (New England Biolabs, cat. no. R0198L) SwaI restriction endonuclease (New England Biolabs, cat. no. R0604L) T4 DNA ligase (New England Biolabs, cat. no. M0202L) XmaI restriction endonuclease (New England Biolabs, cat. no. R0180L) PI-SceI endonuclease (New England Biolabs, cat. no. R0696S) λ-DNA-HindIII Digest (New England Biolabs, cat. no. N3012S) Low-range PFG marker DNA ladder (New England Biolabs, cat. no. NO350S) 2-log DNA ladder (New England Biolabs, cat. no. N3200L) 1-Butanol (Fisher Scientific, cat. no. A399) 2-Propanol (Isopropanol, Fisher Scientific, cat. no. A416) Ammonium acetate (Fisher Scientific, cat. no. A637) Ampicillin (amp; Sigma-Aldrich, cat. no. A9518; see REAGENT SETUP) Calcium chloride dihydrate (CaCl2 , Fisher Scientific, cat. no. C70–500) Cesium chloride (CsCl, Fischer Scientific, cat. no. BP1595-500) Chloramphenicol (chlor; Sigma-Aldrich, cat. no. C0378; see REAGENT SETUP) Chloroform (Fisher Scientific, cat. no. C298–500) Ethidium bromide solution (10 mg ml−1 ; Sigma-Aldrich, cat. no. E1510) ! .. Ethanol (Pharmco-AAPER) EDTA (Sigma-Aldrich, cat. no. E5134) FailSafe PCR System (Epicentre, cat. no. FS99250) Glacial acetic acid (Fisher Scientific, cat. no. A38S) !

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    New England Biolabs xma i
    Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin <t>dIII</t> and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side
    Xma I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

    Journal: Nature Communications

    Article Title: Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4

    doi: 10.1038/s41467-017-01670-6

    Figure Lengend Snippet: Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with Hin dIII and Xma I, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

    Article Snippet: Southern blot analysis For determination of transgene copy number integration by Southern analysis, total nucleic acid was extracted from banana leaf tissue, treated with RNAse A, and 15 μg aliquots of genomic DNA were digested overnight with 20 U of restriction enzyme Hin dIII or Xma I (New England Biolabs) overnight at 37 °C.

    Techniques: Expressing, Transgenic Assay, Southern Blot, Molecular Weight, Marker

    Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.

    Journal: Nature protocols

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

    doi: 10.1038/nprot.2010.131

    Figure Lengend Snippet: Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.

    Article Snippet: Both available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) pSV.RecA vector: available from the laboratory of Nathaniel Heintz, The Rockefeller University, through Judy Walsh ( ) Swiss Webster female mice (Taconic) Bacterial artificial chromosomes (BACs in DH10B host bacteria; Invitrogen Corporation, BACPAC Resources at CHORI or Riken Bioresources Center) PIR1 chemically competent E. coli (Invitrogen, cat. no. C1010-10) PIR2 chemically competent E. coli (Invitrogen, cat. no. C1111-10) MAX efficiency DH5α-competent cells (Invitrogen, cat. no. 18258-012) PCR primers (Invitrogen and Biosynthesis; see REAGENT SETUP) AscI restriction endonuclease (New England Biolabs, cat. no. R0558L) EcoRI restriction endonuclease (New England Biolabs, cat. no. R0101L) MluI restriction endonuclease (New England Biolabs, cat. no. R0198L) SwaI restriction endonuclease (New England Biolabs, cat. no. R0604L) T4 DNA ligase (New England Biolabs, cat. no. M0202L) XmaI restriction endonuclease (New England Biolabs, cat. no. R0180L) PI-SceI endonuclease (New England Biolabs, cat. no. R0696S) λ-DNA-HindIII Digest (New England Biolabs, cat. no. N3012S) Low-range PFG marker DNA ladder (New England Biolabs, cat. no. NO350S) 2-log DNA ladder (New England Biolabs, cat. no. N3200L) 1-Butanol (Fisher Scientific, cat. no. A399) 2-Propanol (Isopropanol, Fisher Scientific, cat. no. A416) Ammonium acetate (Fisher Scientific, cat. no. A637) Ampicillin (amp; Sigma-Aldrich, cat. no. A9518; see REAGENT SETUP) Calcium chloride dihydrate (CaCl2 , Fisher Scientific, cat. no. C70–500) Cesium chloride (CsCl, Fischer Scientific, cat. no. BP1595-500) Chloramphenicol (chlor; Sigma-Aldrich, cat. no. C0378; see REAGENT SETUP) Chloroform (Fisher Scientific, cat. no. C298–500) Ethidium bromide solution (10 mg ml−1 ; Sigma-Aldrich, cat. no. E1510) !

    Techniques: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Journal: The Crispr Journal

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    doi: 10.1089/crispr.2017.0010

    Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Article Snippet: To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

    Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.

    Journal: Nature protocols

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    doi: 10.1038/nprot.2010.129

    Figure Lengend Snippet: Labeling of extracellular tetracysteine-tagged proteins by membrane-impermeant biarsenical dyes. Wide-field images of HEK293 cells transiently transfected with FLNCCPGCCMEP-Myc-V2R-CeFP vasopressin receptor and stained for 10 min with the following: (top row) 2.5 µM sulfo-FlAsH (sFlAsH)-EDT 2 ) and 10 µM EDT after a 30-min preincubation with 5 mM 2-mercaptoethanesulfonate (MES); (middle row) 2.5 µM sFlAsH-EDT 2 and 10 µM EDT after a 30-min preincubation with 0.5 mM tris(carboxyethyl)phosphine (TCEP) and 5 mM MES; (bottom row) 2.5 µM ReAsH-EDT 2 and 5 µM 2,3-dimercaptopropane-sulfonate (DMPS) after a 30-min preincubation with 0.5 mM TCEP and 5 mM MES. Reducing agents were retained during staining and removed with unbound dye by washing with 100 µM DMPS for 10 min before imaging. All procedures were carried out at 37 °C in HBSS/glucose. DIC, differential interference contrast. The white scale bar = 10 µm.

    Article Snippet: Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents.

    Techniques: Labeling, Transfection, Staining, Imaging

    Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.

    Journal: Nature protocols

    Article Title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    doi: 10.1038/nprot.2010.129

    Figure Lengend Snippet: Specific labeling with FlAsH can only be seen after appropriate washing. Confocal images of a human A 2A -adenosine receptor construct with the tetracysteine motif CCPGCC in intracellular loop 3 and CFP at the C-terminus at position Gly-340 (A 2A ) transiently expressed in HEK293 cells. Cells were imaged 48 h after transfection. The top row shows cells after labeling with 500 nM FlAsH according to the protocol without washing (thus, without Steps 11–13). The bottom row shows the same cells 10 min after addition of 250 µM EDT according to the protocol. The left column shows images excited at 430 nm and emission from 470–550 nm (i.e., CFP fluorescence); the middle column shows the same cells excited at 514 nm and emission measured from 530–600 nm (i.e., FlAsH fluorescence). After addition of EDT, the fluorescence intensity of FlAsH-labeled cells was 15- to 20-fold over background of nontransfected neighboring cells. Note that the mere addition of EDT does not represent the full washing procedure and that better background reduction is obtained with the full procedure. The right column represents the transmission images of cells. The white scale bar = 10 µm.

    Article Snippet: Xma I (NEB, 5,000 U, cat. no. R0180S) Sma I (NEB, 2,000 U, cat. no. R0141L) TC-FlAsH (Invitrogen, labeling kit, cat. no. T34561) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents (our FlAsH-EDT2 stock is 1 mM in DMSO; stock solutions are stored at − 20 °C) TC-ReAsH (Invitrogen, labeling kit, cat. no. T34562) Invitrogen is the only commercial supplier, but we have successfully used self-synthesized reagents.

    Techniques: Labeling, Construct, Transfection, Fluorescence, Transmission Assay