Structured Review

Promega ecorv
Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with <t>gDNA</t> obtained from SMCs of 24 subjects. <t>EcoRV</t> digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .
Ecorv, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ecorv/product/Promega
Average 92 stars, based on 55 article reviews
Price from $9.99 to $1999.99
ecorv - by Bioz Stars, 2020-07
92/100 stars

Images

1) Product Images from "A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype"

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114944

Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .
Figure Legend Snippet: Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .

Techniques Used: Polymerase Chain Reaction, Labeling

α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .
Figure Legend Snippet: α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .

Techniques Used: Labeling, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.
Figure Legend Snippet: Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.

Techniques Used: Polymerase Chain Reaction, Labeling, Molecular Weight, Sequencing, Nucleic Acid Electrophoresis

2) Product Images from "Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission"

Article Title: Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

Journal: Scientific Reports

doi: 10.1038/s41598-017-05270-8

Southern blot analysis of the transgenic chickens. Genomic DNA samples were extracted from whole blood samples from wild-type and transgenic chickens (G1 and G2), digested with EcoRV, electrophoresed, blotted, and hybridized with a probe for the 3D8 scFv gene. M, DIG-labeled DNA marker; lane 1, wild-type chicken; lane 2, P-54; lane 3, P-54 offspring; lane 4, N-82; lane 5, N-95; lane 6, N-165; lane 7, N-183; lane 8, P-196; lane 9, N-238.
Figure Legend Snippet: Southern blot analysis of the transgenic chickens. Genomic DNA samples were extracted from whole blood samples from wild-type and transgenic chickens (G1 and G2), digested with EcoRV, electrophoresed, blotted, and hybridized with a probe for the 3D8 scFv gene. M, DIG-labeled DNA marker; lane 1, wild-type chicken; lane 2, P-54; lane 3, P-54 offspring; lane 4, N-82; lane 5, N-95; lane 6, N-165; lane 7, N-183; lane 8, P-196; lane 9, N-238.

Techniques Used: Southern Blot, Transgenic Assay, Labeling, Marker

Related Articles

Clone Assay:

Article Title: Drug-Associated Changes in Amino Acid Residues in Gag p2, p7NC, and p6Gag/p6Pol in Human Immunodeficiency Virus Type 1 (HIV-1) Display a Dominant Effect on Replicative Fitness and Drug Response
Article Snippet: .. To prepare for mutagenesis, a 1.7 kb gag-pol fragment was digested from the pLAI.4-posttherapy gag-pol recombinant virus using the restriction enzymes SphI (NEB) and EcoRV (NEB) and ligated into the pGEM5Zf+ cloning vector (Promega, Madison, WI) that was previously digested with SphI and EcoRV and treated with calf intestinal alkaline phosphatase (CIAP, Promega). .. Following transformation of DH5α cells (Invitrogen, Carlsbad, CA), ten clones were picked, grown overnight at 37°C, and plasmid DNA was extracted using a QIAprep™ Miniprep Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol.

Article Title: Link between the causative genes of holoprosencephaly: Zic2 directly regulates Tgif1 expression
Article Snippet: .. The EcoRV and BglII sites of pGL4 (Promega) were used for the cloning. .. For the Tgif1 in situ hybridization probe, mouse cDNA (RACE-Ready Mouse cDNA, Clontech) was amplified by performing PCR using the following primers: 5′-CTG ATG CTG CAA CAA GAC CCT TCT-3′ and 5′-TAA GCT GTG AGT TTG GCC TGA AGC-3′.

Amplification:

Article Title: Molecular Characterization of a Human Matrix Attachment Region Epigenetic Regulator
Article Snippet: .. Alternatively, the 220 bp PCR amplified product of the AT-rich core region of MAR X-29 was digested completely by EcoRV and purified using the Wizard® SV and PCR clean-up System (Promega), as recommended by the manufacturer, followed by self ligation, giving rise to a series of DNA fragments with increasing copy numbers in various orientations. .. Self-ligated products were separated by gel electrophoresis and fragments in the range from 400 bp to 1 kb were purified using Wizard® SV and PCR clean-up System (Promega) and cloned into pEGFP plasmid.

Agarose Gel Electrophoresis:

Article Title: Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission
Article Snippet: .. Genomic DNA (10 μg) was isolated from whole blood, digested with EcoRV (Promega), resolved on a 1.0% (w/v) agarose gel, and then transferred to a nylon membrane (Roche). ..

Southern Blot:

Article Title: Caspase-2 activation in the absence of PIDDosome formation
Article Snippet: .. Southern blotting and PCR analysis To confirm correct targeting of embryonic stem cells and deletion of the pidd gene in tissues, 20 µg total genomic DNA was digested with the appropriate restriction enzymes (DraIII [New England Biolabs, Inc.] for the 3′ probe and MluI and EcoRV [Promega] for the 5′ probe) overnight. ..

Ligation:

Article Title: Molecular Characterization of a Human Matrix Attachment Region Epigenetic Regulator
Article Snippet: .. Alternatively, the 220 bp PCR amplified product of the AT-rich core region of MAR X-29 was digested completely by EcoRV and purified using the Wizard® SV and PCR clean-up System (Promega), as recommended by the manufacturer, followed by self ligation, giving rise to a series of DNA fragments with increasing copy numbers in various orientations. .. Self-ligated products were separated by gel electrophoresis and fragments in the range from 400 bp to 1 kb were purified using Wizard® SV and PCR clean-up System (Promega) and cloned into pEGFP plasmid.

Mutagenesis:

Article Title: Drug-Associated Changes in Amino Acid Residues in Gag p2, p7NC, and p6Gag/p6Pol in Human Immunodeficiency Virus Type 1 (HIV-1) Display a Dominant Effect on Replicative Fitness and Drug Response
Article Snippet: .. To prepare for mutagenesis, a 1.7 kb gag-pol fragment was digested from the pLAI.4-posttherapy gag-pol recombinant virus using the restriction enzymes SphI (NEB) and EcoRV (NEB) and ligated into the pGEM5Zf+ cloning vector (Promega, Madison, WI) that was previously digested with SphI and EcoRV and treated with calf intestinal alkaline phosphatase (CIAP, Promega). .. Following transformation of DH5α cells (Invitrogen, Carlsbad, CA), ten clones were picked, grown overnight at 37°C, and plasmid DNA was extracted using a QIAprep™ Miniprep Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol.

Isolation:

Article Title: Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission
Article Snippet: .. Genomic DNA (10 μg) was isolated from whole blood, digested with EcoRV (Promega), resolved on a 1.0% (w/v) agarose gel, and then transferred to a nylon membrane (Roche). ..

Purification:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: .. Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Human mast cell leukemia cells (HMC)-1,obtained from a patient with mast cell leukemia and provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) , known to contain only βI and βIII variants of the β-tryptase gene; and Mono Mac-6 cells , a cell line established from a patient with monoblastic leukemia and containing both α- and β- tryptase genes , were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin at 37°C/5% CO2 .

Article Title: Molecular Characterization of a Human Matrix Attachment Region Epigenetic Regulator
Article Snippet: .. Alternatively, the 220 bp PCR amplified product of the AT-rich core region of MAR X-29 was digested completely by EcoRV and purified using the Wizard® SV and PCR clean-up System (Promega), as recommended by the manufacturer, followed by self ligation, giving rise to a series of DNA fragments with increasing copy numbers in various orientations. .. Self-ligated products were separated by gel electrophoresis and fragments in the range from 400 bp to 1 kb were purified using Wizard® SV and PCR clean-up System (Promega) and cloned into pEGFP plasmid.

Polymerase Chain Reaction:

Article Title: Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration
Article Snippet: .. The PCR product was digested with EcoRV and SalI, and inserted into pGL3-promoter vector (Promega) that was first digested with BamH1, blunted and digested again with SalI. .. Constructs for in situ hybridization probes: pBluescript-Krox20 (kind gift from Piotr Topilko), P0 (kind gift from G. Lemke and R. Axel), pBluescript-Oct6 (kind gift from Dies Meijer).

Article Title: Molecular Characterization of a Human Matrix Attachment Region Epigenetic Regulator
Article Snippet: .. Alternatively, the 220 bp PCR amplified product of the AT-rich core region of MAR X-29 was digested completely by EcoRV and purified using the Wizard® SV and PCR clean-up System (Promega), as recommended by the manufacturer, followed by self ligation, giving rise to a series of DNA fragments with increasing copy numbers in various orientations. .. Self-ligated products were separated by gel electrophoresis and fragments in the range from 400 bp to 1 kb were purified using Wizard® SV and PCR clean-up System (Promega) and cloned into pEGFP plasmid.

Article Title: Caspase-2 activation in the absence of PIDDosome formation
Article Snippet: .. Southern blotting and PCR analysis To confirm correct targeting of embryonic stem cells and deletion of the pidd gene in tissues, 20 µg total genomic DNA was digested with the appropriate restriction enzymes (DraIII [New England Biolabs, Inc.] for the 3′ probe and MluI and EcoRV [Promega] for the 5′ probe) overnight. ..

Affinity Purification:

Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype
Article Snippet: .. Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated. .. Human mast cell leukemia cells (HMC)-1,obtained from a patient with mast cell leukemia and provided by Dr. G. Gleich and Dr. J. Butterfield (Mayo Clinic, Rochester, MN) , known to contain only βI and βIII variants of the β-tryptase gene; and Mono Mac-6 cells , a cell line established from a patient with monoblastic leukemia and containing both α- and β- tryptase genes , were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin and streptomycin at 37°C/5% CO2 .

Recombinant:

Article Title: Drug-Associated Changes in Amino Acid Residues in Gag p2, p7NC, and p6Gag/p6Pol in Human Immunodeficiency Virus Type 1 (HIV-1) Display a Dominant Effect on Replicative Fitness and Drug Response
Article Snippet: .. To prepare for mutagenesis, a 1.7 kb gag-pol fragment was digested from the pLAI.4-posttherapy gag-pol recombinant virus using the restriction enzymes SphI (NEB) and EcoRV (NEB) and ligated into the pGEM5Zf+ cloning vector (Promega, Madison, WI) that was previously digested with SphI and EcoRV and treated with calf intestinal alkaline phosphatase (CIAP, Promega). .. Following transformation of DH5α cells (Invitrogen, Carlsbad, CA), ten clones were picked, grown overnight at 37°C, and plasmid DNA was extracted using a QIAprep™ Miniprep Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol.

Plasmid Preparation:

Article Title: Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration
Article Snippet: .. The PCR product was digested with EcoRV and SalI, and inserted into pGL3-promoter vector (Promega) that was first digested with BamH1, blunted and digested again with SalI. .. Constructs for in situ hybridization probes: pBluescript-Krox20 (kind gift from Piotr Topilko), P0 (kind gift from G. Lemke and R. Axel), pBluescript-Oct6 (kind gift from Dies Meijer).

Article Title: Drug-Associated Changes in Amino Acid Residues in Gag p2, p7NC, and p6Gag/p6Pol in Human Immunodeficiency Virus Type 1 (HIV-1) Display a Dominant Effect on Replicative Fitness and Drug Response
Article Snippet: .. To prepare for mutagenesis, a 1.7 kb gag-pol fragment was digested from the pLAI.4-posttherapy gag-pol recombinant virus using the restriction enzymes SphI (NEB) and EcoRV (NEB) and ligated into the pGEM5Zf+ cloning vector (Promega, Madison, WI) that was previously digested with SphI and EcoRV and treated with calf intestinal alkaline phosphatase (CIAP, Promega). .. Following transformation of DH5α cells (Invitrogen, Carlsbad, CA), ten clones were picked, grown overnight at 37°C, and plasmid DNA was extracted using a QIAprep™ Miniprep Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol.

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    Promega ecorv
    Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with <t>gDNA</t> obtained from SMCs of 24 subjects. <t>EcoRV</t> digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .
    Ecorv, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv/product/Promega
    Average 92 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    ecorv - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    93
    Promega ecorv promega restriction enzymes
    Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with <t>gDNA</t> obtained from SMCs of 24 subjects. <t>EcoRV</t> digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .
    Ecorv Promega Restriction Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv promega restriction enzymes/product/Promega
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ecorv promega restriction enzymes - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Polymerase Chain Reaction, Labeling

    α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Labeling, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Polymerase Chain Reaction, Labeling, Molecular Weight, Sequencing, Nucleic Acid Electrophoresis

    Southern blot analysis of the transgenic chickens. Genomic DNA samples were extracted from whole blood samples from wild-type and transgenic chickens (G1 and G2), digested with EcoRV, electrophoresed, blotted, and hybridized with a probe for the 3D8 scFv gene. M, DIG-labeled DNA marker; lane 1, wild-type chicken; lane 2, P-54; lane 3, P-54 offspring; lane 4, N-82; lane 5, N-95; lane 6, N-165; lane 7, N-183; lane 8, P-196; lane 9, N-238.

    Journal: Scientific Reports

    Article Title: Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

    doi: 10.1038/s41598-017-05270-8

    Figure Lengend Snippet: Southern blot analysis of the transgenic chickens. Genomic DNA samples were extracted from whole blood samples from wild-type and transgenic chickens (G1 and G2), digested with EcoRV, electrophoresed, blotted, and hybridized with a probe for the 3D8 scFv gene. M, DIG-labeled DNA marker; lane 1, wild-type chicken; lane 2, P-54; lane 3, P-54 offspring; lane 4, N-82; lane 5, N-95; lane 6, N-165; lane 7, N-183; lane 8, P-196; lane 9, N-238.

    Article Snippet: Genomic DNA (10 μg) was isolated from whole blood, digested with EcoRV (Promega), resolved on a 1.0% (w/v) agarose gel, and then transferred to a nylon membrane (Roche).

    Techniques: Southern Blot, Transgenic Assay, Labeling, Marker