hand1 locus  (New England Biolabs)


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    New England Biolabs hand1 locus
    KpnI HF
    KpnI HF 20 000 units
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    Average 84 stars, based on 17338 article reviews
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    hand1 locus - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Impaired labyrinth formation prevents the establishment of the maternal-fetal interface in conditional Hand1-deficient mice"

    Article Title: Impaired labyrinth formation prevents the establishment of the maternal-fetal interface in conditional Hand1-deficient mice

    Journal: bioRxiv

    doi: 10.1101/2020.09.02.280354

    (A) Relative Plgf expression was significantly increased in the Nkx2.5 cre ; Hand1 A126fs/+ labyrinths verses littermate controls. (B-E) Relative gene expression of VegFa, VegFb, Angpt1, and Angpt2 were not significantly different between Nkx2.5 cre ; Hand1 A126fs/+ and Nkx2.5 cre ;Hand1 +/+ placental labyrinthine tissue at E10.5, although Angpt2 trended higher in the conditional Han1 knockouts. Bars indicate mean ± SEM.
    Figure Legend Snippet: (A) Relative Plgf expression was significantly increased in the Nkx2.5 cre ; Hand1 A126fs/+ labyrinths verses littermate controls. (B-E) Relative gene expression of VegFa, VegFb, Angpt1, and Angpt2 were not significantly different between Nkx2.5 cre ; Hand1 A126fs/+ and Nkx2.5 cre ;Hand1 +/+ placental labyrinthine tissue at E10.5, although Angpt2 trended higher in the conditional Han1 knockouts. Bars indicate mean ± SEM.

    Techniques Used: Expressing

    (B,D,F) Fetal blood vessel counts were significantly reduced in the Nkx2.5 cre ;Hand1 A126fs/+ labyrinth at E12.5 compared to littermate controls (31.8±3.4 vs 2.6±1.2, p=0.001). (A,C,E) While not significantly different at E10.5, vessel counts trended downward at E10.5 (13.6±1.4 vs 7.4±3.4, p=0.088) with decreased branching and lack of contact with maternal blood spaces. Vessels were stained with CD-31 for fetal endothelium (brown). Arrowheads indicate fetal vessels, while asterisks mark maternal blood spaces. N= 4-6 litters, bars indicate mean ± SEM. ** p
    Figure Legend Snippet: (B,D,F) Fetal blood vessel counts were significantly reduced in the Nkx2.5 cre ;Hand1 A126fs/+ labyrinth at E12.5 compared to littermate controls (31.8±3.4 vs 2.6±1.2, p=0.001). (A,C,E) While not significantly different at E10.5, vessel counts trended downward at E10.5 (13.6±1.4 vs 7.4±3.4, p=0.088) with decreased branching and lack of contact with maternal blood spaces. Vessels were stained with CD-31 for fetal endothelium (brown). Arrowheads indicate fetal vessels, while asterisks mark maternal blood spaces. N= 4-6 litters, bars indicate mean ± SEM. ** p

    Techniques Used: Staining

    Nkx2.5 cre ;Hand1 +/+ panels A-C, Nkx2.5 cre ;Hand1 A126fs/+ panels D-F. Left column E9.5, middle E10.5, right E12.5. CD31-positive (stained red) fetal blood vessels are not present at E9.5 (A, D) in the chorionic plate for either genotype but both contain a layer of CK-7-positive (green) trophoblasts. Conditional Hand1 knockouts (E-F) show a disruption in syncytium with only sinusoidal trophoblast giant cells (arrowhead) lining the maternal blood spaces, while control littermates (B-C) have both a defined syncytium and labyrinthine fetal blood vessels.CD31-positive fetal endothelium (asterisk) is absent in conditional Hand1 knockouts at E10.5 and E12.5.. Red CD-31 (fetal endothelium), green Cytokeratin-7 (trophoblast), blue is DAPI.40X representative images.
    Figure Legend Snippet: Nkx2.5 cre ;Hand1 +/+ panels A-C, Nkx2.5 cre ;Hand1 A126fs/+ panels D-F. Left column E9.5, middle E10.5, right E12.5. CD31-positive (stained red) fetal blood vessels are not present at E9.5 (A, D) in the chorionic plate for either genotype but both contain a layer of CK-7-positive (green) trophoblasts. Conditional Hand1 knockouts (E-F) show a disruption in syncytium with only sinusoidal trophoblast giant cells (arrowhead) lining the maternal blood spaces, while control littermates (B-C) have both a defined syncytium and labyrinthine fetal blood vessels.CD31-positive fetal endothelium (asterisk) is absent in conditional Hand1 knockouts at E10.5 and E12.5.. Red CD-31 (fetal endothelium), green Cytokeratin-7 (trophoblast), blue is DAPI.40X representative images.

    Techniques Used: Staining

    Nkx2.5 cre ;Hand1 A126FS/+ show increased fetal demise. Graph shows percentages of live Nkx2.5 cre ;Hand1 +/+ (white), Nkx2.5 cre ;Hand1 A126fs/+ (grey), and resorbed (black) fetuses at E10.5, E12.5, and E14.5. No live Nkx2.5 cre ;Hand1 A126fs/+ fetuses were retrieved at E14.5. N = 4-8 litters.
    Figure Legend Snippet: Nkx2.5 cre ;Hand1 A126FS/+ show increased fetal demise. Graph shows percentages of live Nkx2.5 cre ;Hand1 +/+ (white), Nkx2.5 cre ;Hand1 A126fs/+ (grey), and resorbed (black) fetuses at E10.5, E12.5, and E14.5. No live Nkx2.5 cre ;Hand1 A126fs/+ fetuses were retrieved at E14.5. N = 4-8 litters.

    Techniques Used:

    Labyrinthine morphogenesis is altered in Nkx2.5cre;Hand1A126fs/+ placentas at E9.5. (A) Control littermate placentas exhibit invagination of the chorion and development of an early labyrinth at E9.5. (B). In contrast, Nkx2.5cre;Hand1A126fs/+ placentas lack a chorionic plate and only have a disorganized layer of trophoblast giant cells surrounding the amniotic cavity. (C, D). Yolk sac morphology is altered in Nkx2.5 cre ;Hand1 A126fs/+ fetuses at E9.5. (C). Normal littermates show adjacent chorion and yolk sac (arrow), while (D) Nkx2.5 cre ;Hand1 A126fs/+ placentas lack a chorionic layer and yolk sacs are detached from the trophoblast layer separated by large maternal blood spaces (indicated by asterisks). (A,B) immunostained for Hand1. (C,D) stained with hematoxylin and eosin
    Figure Legend Snippet: Labyrinthine morphogenesis is altered in Nkx2.5cre;Hand1A126fs/+ placentas at E9.5. (A) Control littermate placentas exhibit invagination of the chorion and development of an early labyrinth at E9.5. (B). In contrast, Nkx2.5cre;Hand1A126fs/+ placentas lack a chorionic plate and only have a disorganized layer of trophoblast giant cells surrounding the amniotic cavity. (C, D). Yolk sac morphology is altered in Nkx2.5 cre ;Hand1 A126fs/+ fetuses at E9.5. (C). Normal littermates show adjacent chorion and yolk sac (arrow), while (D) Nkx2.5 cre ;Hand1 A126fs/+ placentas lack a chorionic layer and yolk sacs are detached from the trophoblast layer separated by large maternal blood spaces (indicated by asterisks). (A,B) immunostained for Hand1. (C,D) stained with hematoxylin and eosin

    Techniques Used: Staining

    2) Product Images from "IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production"

    Article Title: IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.03.002

    IQGAP1 Interacts with the Nucleocapsid and p6 Domains of HIV-1 Gag (A) IQGAP1 interacts with the HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) together with DNAs expressing GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to pNL4–3.Luc.R-E-). Cell lysates were subjected to immunoprecipitation using anti-GFP antibody. GFP, GFP-IQGAP1, and Gag were detected using western blot. Shown are representative western blots from three independent experiments (n = 3). (B) IQGAP1 interacts with unmyristylated HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (Gag WT; 1 µg) or with an HIV-1 genome encoding a Gag mutant that cannot be myristylated (Gag G2A; 1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag WT or Gag G2A) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (C) Schematic presentation of the Rev-independent flag-tagged Gag domains proteins used for this experiment. (D) The nucleocapsid domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with DNAs encoding GFP or GFP-IQGAP1 together with 1 µg of the indicated Gag constructs from (C) at a molar ratio of 3 (GFP or GFP-IQGAP1 to HIV-1 Gag constructs) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (E) The p6 domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with the GagDNC construct (C) (1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to GagΔDNC) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).
    Figure Legend Snippet: IQGAP1 Interacts with the Nucleocapsid and p6 Domains of HIV-1 Gag (A) IQGAP1 interacts with the HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) together with DNAs expressing GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to pNL4–3.Luc.R-E-). Cell lysates were subjected to immunoprecipitation using anti-GFP antibody. GFP, GFP-IQGAP1, and Gag were detected using western blot. Shown are representative western blots from three independent experiments (n = 3). (B) IQGAP1 interacts with unmyristylated HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (Gag WT; 1 µg) or with an HIV-1 genome encoding a Gag mutant that cannot be myristylated (Gag G2A; 1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag WT or Gag G2A) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (C) Schematic presentation of the Rev-independent flag-tagged Gag domains proteins used for this experiment. (D) The nucleocapsid domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with DNAs encoding GFP or GFP-IQGAP1 together with 1 µg of the indicated Gag constructs from (C) at a molar ratio of 3 (GFP or GFP-IQGAP1 to HIV-1 Gag constructs) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (E) The p6 domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with the GagDNC construct (C) (1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to GagΔDNC) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Construct

    The C Term inus Region of HIV-1 Gag Is Required for IQGAP1 Regulation of Viral Release Independently of Gag L Domains (A) Schematic presentation of Rev-independent flag-tagged Gag domains proteins used for this experiment. (B and C) Replacement of the C terminus region of Gag with the GCN4 leucine zipper abolishes IQGAP1 regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag-flag (B) or GagZ-flag (C) constructs (0.5 µg) with increasing amounts of DNA encoding GFP-IQGAP1 or GFP at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the culture supernatants were purified. CA levels in pelleted virions and intracellular GFP-IQGAP1 and Gag levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Shown are representative western blots from three independent experiments (n = 3). (D and E) Either the p6 or the nucleocapsid domains in Gag are sufficient for IQGAP1 negative regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent GagΔp6-flag (D) or GagDNC-flag (E) DNAs (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNA and processed as in (B). Shown are representative western blots from three independent experiments (n = 3). (F) Schematic presentation of Rev-independent flag-tagged Gag mutant proteins used for this research. (G–J) IQGAP1 overexpression reduces HIV-1 Gag VLPs lacking the ability to recruit the cellular ESCRT machinery. HEK293T cells were co-transfected with DNA constructs from (F) (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the supernatants were isolated. CA levels in pelleted virions and cellular GFP-IQGAP1 and Gag expression levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Cells were transfected with (G) wild-type, (H) PTAPmut, (I) LYPXnLmut, (J) and PTAP/LYPXnLmut Gag expression constructs. Shown are representative western blots from three independent experiments (n = 3).
    Figure Legend Snippet: The C Term inus Region of HIV-1 Gag Is Required for IQGAP1 Regulation of Viral Release Independently of Gag L Domains (A) Schematic presentation of Rev-independent flag-tagged Gag domains proteins used for this experiment. (B and C) Replacement of the C terminus region of Gag with the GCN4 leucine zipper abolishes IQGAP1 regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag-flag (B) or GagZ-flag (C) constructs (0.5 µg) with increasing amounts of DNA encoding GFP-IQGAP1 or GFP at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the culture supernatants were purified. CA levels in pelleted virions and intracellular GFP-IQGAP1 and Gag levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Shown are representative western blots from three independent experiments (n = 3). (D and E) Either the p6 or the nucleocapsid domains in Gag are sufficient for IQGAP1 negative regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent GagΔp6-flag (D) or GagDNC-flag (E) DNAs (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNA and processed as in (B). Shown are representative western blots from three independent experiments (n = 3). (F) Schematic presentation of Rev-independent flag-tagged Gag mutant proteins used for this research. (G–J) IQGAP1 overexpression reduces HIV-1 Gag VLPs lacking the ability to recruit the cellular ESCRT machinery. HEK293T cells were co-transfected with DNA constructs from (F) (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the supernatants were isolated. CA levels in pelleted virions and cellular GFP-IQGAP1 and Gag expression levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Cells were transfected with (G) wild-type, (H) PTAPmut, (I) LYPXnLmut, (J) and PTAP/LYPXnLmut Gag expression constructs. Shown are representative western blots from three independent experiments (n = 3).

    Techniques Used: Transfection, Construct, Plasmid Preparation, Purification, Western Blot, Mutagenesis, Over Expression, Isolation, Expressing

    3) Product Images from "Impaired labyrinth formation prevents the establishment of the maternal-fetal interface in conditional Hand1-deficient mice"

    Article Title: Impaired labyrinth formation prevents the establishment of the maternal-fetal interface in conditional Hand1-deficient mice

    Journal: bioRxiv

    doi: 10.1101/2020.09.02.280354

    (A) Relative Plgf expression was significantly increased in the Nkx2.5 cre ; Hand1 A126fs/+ labyrinths verses littermate controls. (B-E) Relative gene expression of VegFa, VegFb, Angpt1, and Angpt2 were not significantly different between Nkx2.5 cre ; Hand1 A126fs/+ and Nkx2.5 cre ;Hand1 +/+ placental labyrinthine tissue at E10.5, although Angpt2 trended higher in the conditional Han1 knockouts. Bars indicate mean ± SEM.
    Figure Legend Snippet: (A) Relative Plgf expression was significantly increased in the Nkx2.5 cre ; Hand1 A126fs/+ labyrinths verses littermate controls. (B-E) Relative gene expression of VegFa, VegFb, Angpt1, and Angpt2 were not significantly different between Nkx2.5 cre ; Hand1 A126fs/+ and Nkx2.5 cre ;Hand1 +/+ placental labyrinthine tissue at E10.5, although Angpt2 trended higher in the conditional Han1 knockouts. Bars indicate mean ± SEM.

    Techniques Used: Expressing

    (B,D,F) Fetal blood vessel counts were significantly reduced in the Nkx2.5 cre ;Hand1 A126fs/+ labyrinth at E12.5 compared to littermate controls (31.8±3.4 vs 2.6±1.2, p=0.001). (A,C,E) While not significantly different at E10.5, vessel counts trended downward at E10.5 (13.6±1.4 vs 7.4±3.4, p=0.088) with decreased branching and lack of contact with maternal blood spaces. Vessels were stained with CD-31 for fetal endothelium (brown). Arrowheads indicate fetal vessels, while asterisks mark maternal blood spaces. N= 4-6 litters, bars indicate mean ± SEM. ** p
    Figure Legend Snippet: (B,D,F) Fetal blood vessel counts were significantly reduced in the Nkx2.5 cre ;Hand1 A126fs/+ labyrinth at E12.5 compared to littermate controls (31.8±3.4 vs 2.6±1.2, p=0.001). (A,C,E) While not significantly different at E10.5, vessel counts trended downward at E10.5 (13.6±1.4 vs 7.4±3.4, p=0.088) with decreased branching and lack of contact with maternal blood spaces. Vessels were stained with CD-31 for fetal endothelium (brown). Arrowheads indicate fetal vessels, while asterisks mark maternal blood spaces. N= 4-6 litters, bars indicate mean ± SEM. ** p

    Techniques Used: Staining

    Nkx2.5 cre ;Hand1 +/+ panels A-C, Nkx2.5 cre ;Hand1 A126fs/+ panels D-F. Left column E9.5, middle E10.5, right E12.5. CD31-positive (stained red) fetal blood vessels are not present at E9.5 (A, D) in the chorionic plate for either genotype but both contain a layer of CK-7-positive (green) trophoblasts. Conditional Hand1 knockouts (E-F) show a disruption in syncytium with only sinusoidal trophoblast giant cells (arrowhead) lining the maternal blood spaces, while control littermates (B-C) have both a defined syncytium and labyrinthine fetal blood vessels.CD31-positive fetal endothelium (asterisk) is absent in conditional Hand1 knockouts at E10.5 and E12.5.. Red CD-31 (fetal endothelium), green Cytokeratin-7 (trophoblast), blue is DAPI.40X representative images.
    Figure Legend Snippet: Nkx2.5 cre ;Hand1 +/+ panels A-C, Nkx2.5 cre ;Hand1 A126fs/+ panels D-F. Left column E9.5, middle E10.5, right E12.5. CD31-positive (stained red) fetal blood vessels are not present at E9.5 (A, D) in the chorionic plate for either genotype but both contain a layer of CK-7-positive (green) trophoblasts. Conditional Hand1 knockouts (E-F) show a disruption in syncytium with only sinusoidal trophoblast giant cells (arrowhead) lining the maternal blood spaces, while control littermates (B-C) have both a defined syncytium and labyrinthine fetal blood vessels.CD31-positive fetal endothelium (asterisk) is absent in conditional Hand1 knockouts at E10.5 and E12.5.. Red CD-31 (fetal endothelium), green Cytokeratin-7 (trophoblast), blue is DAPI.40X representative images.

    Techniques Used: Staining

    Nkx2.5 cre ;Hand1 A126FS/+ show increased fetal demise. Graph shows percentages of live Nkx2.5 cre ;Hand1 +/+ (white), Nkx2.5 cre ;Hand1 A126fs/+ (grey), and resorbed (black) fetuses at E10.5, E12.5, and E14.5. No live Nkx2.5 cre ;Hand1 A126fs/+ fetuses were retrieved at E14.5. N = 4-8 litters.
    Figure Legend Snippet: Nkx2.5 cre ;Hand1 A126FS/+ show increased fetal demise. Graph shows percentages of live Nkx2.5 cre ;Hand1 +/+ (white), Nkx2.5 cre ;Hand1 A126fs/+ (grey), and resorbed (black) fetuses at E10.5, E12.5, and E14.5. No live Nkx2.5 cre ;Hand1 A126fs/+ fetuses were retrieved at E14.5. N = 4-8 litters.

    Techniques Used:

    Labyrinthine morphogenesis is altered in Nkx2.5cre;Hand1A126fs/+ placentas at E9.5. (A) Control littermate placentas exhibit invagination of the chorion and development of an early labyrinth at E9.5. (B). In contrast, Nkx2.5cre;Hand1A126fs/+ placentas lack a chorionic plate and only have a disorganized layer of trophoblast giant cells surrounding the amniotic cavity. (C, D). Yolk sac morphology is altered in Nkx2.5 cre ;Hand1 A126fs/+ fetuses at E9.5. (C). Normal littermates show adjacent chorion and yolk sac (arrow), while (D) Nkx2.5 cre ;Hand1 A126fs/+ placentas lack a chorionic layer and yolk sacs are detached from the trophoblast layer separated by large maternal blood spaces (indicated by asterisks). (A,B) immunostained for Hand1. (C,D) stained with hematoxylin and eosin
    Figure Legend Snippet: Labyrinthine morphogenesis is altered in Nkx2.5cre;Hand1A126fs/+ placentas at E9.5. (A) Control littermate placentas exhibit invagination of the chorion and development of an early labyrinth at E9.5. (B). In contrast, Nkx2.5cre;Hand1A126fs/+ placentas lack a chorionic plate and only have a disorganized layer of trophoblast giant cells surrounding the amniotic cavity. (C, D). Yolk sac morphology is altered in Nkx2.5 cre ;Hand1 A126fs/+ fetuses at E9.5. (C). Normal littermates show adjacent chorion and yolk sac (arrow), while (D) Nkx2.5 cre ;Hand1 A126fs/+ placentas lack a chorionic layer and yolk sacs are detached from the trophoblast layer separated by large maternal blood spaces (indicated by asterisks). (A,B) immunostained for Hand1. (C,D) stained with hematoxylin and eosin

    Techniques Used: Staining

    Related Articles

    Amplification:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Subcloning:

    Article Title: High-performance chemical- and light-inducible recombinases in mammalian cells and mice
    Article Snippet: .. A similar process was followed as before with the C-terminal CID and LID sub-cloning vectors; however, Kpn I-HF and Eco RI-HF restriction digests (New England Biolabs) were carried out rather than with Mlu I and Bsp EI. .. To swap between CID and LID domains, sequenced fragments were gel isolated from sequenced vectors using Mlu I/Bsp EI or Kpn I-HF/Eco RI-HF and inserted into corresponding CID or LID sub-cloning vectors.

    Purification:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Polymerase Chain Reaction:

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders
    Article Snippet: .. The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA). .. Cleavage fragments from PCR products of wild type Dnd1 (180 bp and 380 bp) and unchanged 560 bp ter allele products were separated by electrophoresis in a 1.5% agarose gel in 1× TBE buffer.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: The genome-wide transcriptional response to varying RpoS levels in Escherichia coli K-12
    Article Snippet: .. PCR products or oligonucleotides were mixed with pLFX digested with KpnI-HF and EcoRI-HF, and assembled according to the manufacturers’ instructions. .. Mixtures were cloned into strain BW23473, transformants were miniprepped, and inserts were verified by Sanger sequencing. lacZ fusion plasmids were integrated into strain DMS2564 with helper plasmid pPFINT ( ).

    Generated:

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Positron Emission Tomography:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Expressing:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Sequencing:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Plasmid Preparation:

    Article Title: Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase
    Article Snippet: .. The EGFP fragment was then excised from the pEGFP-N2 parent vector by enzyme digestion with Kpn I-HF and Not I-HF. .. The vector was gel-purified with QIAquick gel extraction kit (Qiagen) and the fragments of mCherry, sYFP2, or mTurquoise2 ( ) were ligated using T4 DNA ligase (Thermo Fisher Scientific) into the pEGFP-N2 vector, which was digested with Kpn I and Not I.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

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    New England Biolabs fau i
    Fau I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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