bsa  (New England Biolabs)


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    BSA-Molecular Biology Grade
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    Structured Review

    New England Biolabs bsa
    LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on <t>BSA,</t> LN-2, <t>TSP-1,</t> and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P

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    1) Product Images from "Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination"

    Article Title: Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200304154

    LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P
    Figure Legend Snippet: LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P

    Techniques Used: Activity Assay, Dominant Negative Mutation, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P
    Figure Legend Snippet: LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P

    Techniques Used: In Vitro, Purification, Isolation, Cell Culture, Staining

    Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P
    Figure Legend Snippet: Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P

    Techniques Used: Blocking Assay, Staining, Infection, Plasmid Preparation, Dominant Negative Mutation, Double Staining, Hemagglutination Assay, Incubation, Expressing

    2) Product Images from "The fragile X mental retardation protein inhibits translation via interacting with mRNA"

    Article Title: The fragile X mental retardation protein inhibits translation via interacting with mRNA

    Journal:

    doi:

    Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P < 0.001). The number of experiments for each treatment is also depicted at the bottom. The asterisks indicate P values (***, P < 0.001) in comparison to the yield from the mock-treated reaction. ( B ) SDS–PAGE analysis of the translation inhibition caused by a high concentration of FMRP. FMRP concentration in the reaction was 250 nM, as depicted at the top of the corresponding lane. ( C ) Coomassie Blue staining of a SDS–PAGE gel of FMRP and BSA used in the translation reaction. The amount of protein loaded is indicated at the top of the corresponding lane. ( D ) Recombinant FMRP remains intact during translation. Translation mix containing various amounts of Flag–FMRP, as indicated at the top of the corresponding lanes, was subjected to SDS–PAGE immunoblotting at the end of the reaction by anti-Flag antibody.
    Figure Legend Snippet: Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P < 0.001). The number of experiments for each treatment is also depicted at the bottom. The asterisks indicate P values (***, P < 0.001) in comparison to the yield from the mock-treated reaction. ( B ) SDS–PAGE analysis of the translation inhibition caused by a high concentration of FMRP. FMRP concentration in the reaction was 250 nM, as depicted at the top of the corresponding lane. ( C ) Coomassie Blue staining of a SDS–PAGE gel of FMRP and BSA used in the translation reaction. The amount of protein loaded is indicated at the top of the corresponding lane. ( D ) Recombinant FMRP remains intact during translation. Translation mix containing various amounts of Flag–FMRP, as indicated at the top of the corresponding lanes, was subjected to SDS–PAGE immunoblotting at the end of the reaction by anti-Flag antibody.

    Techniques Used: SDS Page, Inhibition, Concentration Assay, Staining, Recombinant

    3) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.
    Figure Legend Snippet: Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.

    Techniques Used: Affinity Purification, Purification, SDS Page, Staining, Marker, Incubation, Negative Control, Western Blot

    4) Product Images from "Bicistronic DNA display for in vitro selection of Fab fragments"

    Article Title: Bicistronic DNA display for in vitro selection of Fab fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp776

    Scheme of in vitro selection of Fab fragments using DNA display. Step 1: The template DNA has two ORFs (a streptavidin-fused H-chain gene and an L-chain gene), one T7 promoter (T7) and two ribosomal binding sites (SD). The DNA library is biotinylated through a photo-cleavable linker and compartmentalized in water-in-oil emulsions containing an in vitro transcription/translation system. Step 2: In each micelle, a streptavidin-fused H-chain and an L-chain are expressed, forming a Fab fragment and linked to the corresponding DNA via streptavidin-biotin linkage. Step 3: DNA-displayed Fab fragments are recovered from the emulsion and subjected to in vitro antigen selection. Step 4: Selected DNA-displayed Fab fragments are exposed to UV irradiation at > 300 nm to cleave the DNA for elution. Step 5: Selected DNA is amplified by PCR with biotinylated primers to make templates for the next round of selection. Step 6: After a suitable number of rounds of selection, the selected DNA is cloned and sequenced to identify the selected Fab fragments.
    Figure Legend Snippet: Scheme of in vitro selection of Fab fragments using DNA display. Step 1: The template DNA has two ORFs (a streptavidin-fused H-chain gene and an L-chain gene), one T7 promoter (T7) and two ribosomal binding sites (SD). The DNA library is biotinylated through a photo-cleavable linker and compartmentalized in water-in-oil emulsions containing an in vitro transcription/translation system. Step 2: In each micelle, a streptavidin-fused H-chain and an L-chain are expressed, forming a Fab fragment and linked to the corresponding DNA via streptavidin-biotin linkage. Step 3: DNA-displayed Fab fragments are recovered from the emulsion and subjected to in vitro antigen selection. Step 4: Selected DNA-displayed Fab fragments are exposed to UV irradiation at > 300 nm to cleave the DNA for elution. Step 5: Selected DNA is amplified by PCR with biotinylated primers to make templates for the next round of selection. Step 6: After a suitable number of rounds of selection, the selected DNA is cloned and sequenced to identify the selected Fab fragments.

    Techniques Used: In Vitro, Selection, Binding Assay, Irradiation, Amplification, Polymerase Chain Reaction, Clone Assay

    5) Product Images from "Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase"

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl060

    Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).
    Figure Legend Snippet: Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).

    Techniques Used: Purification, Flow Cytometry

    6) Product Images from "The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1"

    Article Title: The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr040

    The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P
    Figure Legend Snippet: The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P

    Techniques Used: Mutagenesis, Irradiation, In Vitro, Kinase Assay, Recombinant, Incubation, Autoradiography, Plasmid Preparation, Quantitative RT-PCR, Expressing, Hemagglutination Assay, Chromatin Immunoprecipitation

    7) Product Images from "Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks"

    Article Title: Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks

    Journal: Nature cell biology

    doi: 10.1038/ncb3643

    Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P
    Figure Legend Snippet: Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P

    Techniques Used: Sequencing, Expressing, Injection, Labeling, Incubation, Single Molecule Counting, Two Tailed Test

    8) Product Images from "Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth"

    Article Title: Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth

    Journal:

    doi: 10.1128/JB.01506-10

    HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared
    Figure Legend Snippet: HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared

    Techniques Used: Far Western Blot, Purification, Negative Control, Immunostaining

    9) Product Images from "Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies"

    Article Title: Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18050937

    Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).
    Figure Legend Snippet: Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).

    Techniques Used: Binding Assay, Activity Assay, Recombinant, SDS Page, Purification, Produced, Flow Cytometry, Fluorescence, Negative Control, Magnetic Beads

    10) Product Images from "Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair"

    Article Title: Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201005110

    Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.
    Figure Legend Snippet: Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.

    Techniques Used: Functional Assay, Recombinase Polymerase Amplification, Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing, SDS Page, Western Blot, Quantitation Assay, Purification, Recombinant

    11) Product Images from "Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA"

    Article Title: Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2010.07292.x

    CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).
    Figure Legend Snippet: CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

    12) Product Images from "Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion"

    Article Title: Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2011.07927.x

    Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .
    Figure Legend Snippet: Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .

    Techniques Used: SDS Page, Selection, Positive Control, Mutagenesis, Negative Control, Activated Clotting Time Assay, Cell Culture, Staining

    13) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.
    Figure Legend Snippet: Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

    Techniques Used: Plasmid Preparation, Clone Assay, Marker, Amplification, Functional Assay, Sequencing

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    Amplification:

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    Synthesized:

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    ChIA Pet Assay:

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    Construct:

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    Real-time Polymerase Chain Reaction:

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    Microarray:

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    Incubation:

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    Formalin-fixed Paraffin-Embedded:

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    Footprinting:

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    Expressing:

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    Modification:

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    Western Blot:

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    Transformation Assay:

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    Hybridization:

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    Electroporation:

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
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    Flow Cytometry:

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    Ligation:

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Following digestion, MboI enzyme was heat inactivated by incubating the nuclei at 62°C for 20 min. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52 μL of fill-in master mix, containing 37.5 μL of 0.4 mM biotin-dATP (Invitrogen, 19524016), 1.5 μL of 10 mM dCTP (Invitrogen, 18253013), 1.5 μL of 10 mM dGTP (Invitrogen, 18254011), 1.5 μL of 10 mM dTTP (Invitrogen, 18255018), and 10 μL of 5 U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210), was added and the tubes were incubated at 37°C for 1 hour with rotation. .. Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation. .. After proximity ligation, nuclei were pelleted by centrifugation at 2500 r cf. for 5 minutes and resuspended in 1 mL of ChIP sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS with protease inhibitor).

    Protease Inhibitor:

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: Cells were pelleted and resuspended in 500 µl cold Hi-C lysis buffer (10 mM Tris-HCl pH8, 10 mM NaCl, 0.2% Igepal CA-630, and 1× Protease Inhibitor (Roche 11873580001) and incubated on ice for 1 h. Nuclei were pelleted at 2500 rcf for 5 min at 4°C, resuspended in 100 µl 0. .. This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202).

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Cross-linked cell pellets were thawed on ice, resuspended in 800 μL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% IGEPAL CA-630 with 1× cOmplete protease inhibitor (Roche, 11697498001)), and incubated at 4°C for 30 minutes with rotation. .. Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation.

    Dissection:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: Tumor and non-tumor areas were separately microdissected from sections of archival paraffin-embedded tissue using a dissection microscope. .. BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    SDS Page:

    Article Title: Specific DNA-binding by Apicomplexan AP2 transcription factors
    Article Snippet: Purity was verified by silver stain SDS/PAGE and Western blot analysis with an anti-GST antibody (Invitrogen). .. Purified proteins were diluted to a final concentration of 100–500 nM in PBS, 2% (wt/vol) milk, 51.3 ng/μl salmon testes DNA (Sigma), 0.2 μg/μl BSA (New England Biolabs), and incubated for 1 h at 20°C.

    Generated:

    Article Title: Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes
    Article Snippet: BNA primer hybridization and mRNA reverse transcription were performed o/n at 42 °C using 200 nM of BNA primer (IDT, Leuven Belgium), 20 U/µl RevertAid H minus M-MuLV reverse transcriptase in 1x M-MuLV reaction buffer (ThermoFisher Scientific, Waltham, MA, USA), 1 U/µl RiboLock RNase Inhibitor (ThermoFisher Scientific, Waltham, MA, USA), 500 µM dNTP and 0.2 µg/µl BSA (New England Biolabs, Ipswich, MA, USA). .. BNA primer hybridization and mRNA reverse transcription were performed o/n at 42 °C using 200 nM of BNA primer (IDT, Leuven Belgium), 20 U/µl RevertAid H minus M-MuLV reverse transcriptase in 1x M-MuLV reaction buffer (ThermoFisher Scientific, Waltham, MA, USA), 1 U/µl RiboLock RNase Inhibitor (ThermoFisher Scientific, Waltham, MA, USA), 500 µM dNTP and 0.2 µg/µl BSA (New England Biolabs, Ipswich, MA, USA).

    Polymerase Chain Reaction:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: Mutations in BAP1 (all exons), BRAF (exon 15), and HRAS (exons 1 and 2) were determined by direct sequencing using previously described primers., The PCR reaction conditions were 0.25 mM dNTPs, 0.4. .. BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Taken together, these findings suggest that SauCas9 may be an attractive alternative or complement to SpyCas9 and could possibly be leveraged for future biotechnological and therapeutic applications. .. S. pyogenes Cas9 (# M0386M), EnGen sgRNA Synthesis Kit, S. pyogenes (# E3322S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S), 2× RNA Loading Dye (# B0363S), Nucleoside Digestion Mix (M0649S), Q5 Hot Start High-Fidelity 2× Master Mix (# M0494L), Monarch PCR & DNA Cleanup Kit (# T1030L), Proteinase K (# P8107S), Shrimp Alkaline Phosphatase (# M0371S), T4 Polynucleotide Kinase (# M0201S), Streptavidin Magnetic Beads (# S1420S), and NEBuffer 3.1 (# B7203S) with a 1× composition of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 at 25°C were all from New England Biolabs. .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques.

    Article Title: Association between two ?-opioid receptor gene (OPRM1) haplotype blocks and drug or alcohol dependence
    Article Snippet: All other SNPs were genotyped using the TaqMan assay in a 384-well microplate format. .. Briefly, 1 ng of DNA was amplified in a final volume of 2 μl containing 0.05 μl of 20× (or 0.025 μl of 40×) MGB probes and primers, 1 μl of 2× TaqMan Universal PCR Master Mix, and 0.004 μl of 100× BSA (New England Biolabs Inc., Beverly, MA). .. Amplification conditions were 95°C for 10 min, followed by 60 cycles of 92°C for 15 s and 60°C for 1 min. Allelic discrimination was performed using the ABI PRISM® 7900 Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA).

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: Cosmid and plasmid DNAs were extracted from E. coli by the alkaline method ( ). .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics. .. Digoxigenin-DNA labeling (digoxigenin dUTP), hybridization, washings, and detection were performed according to the recommendations of the manufacturer (Roche).

    DNA Sequencing:

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics. .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Mutation identification in a canine model of X-linked ectodermal dysplasia
    Article Snippet: Paragraph title: cDNA synthesis and RT-PCR ... Total RNA was extracted from skin and kidney from normal and affected dogs using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. cDNA was synthesized in a 50-μl reaction containing 10 μg total RNA 1 × first-strand synthesis buffer (Invitrogen, Carlsbad, CA), 0.1 mM DTT (Invitrogen), 8 mM dNTPs (Promega, Madison, WI), 0.2 μg/μl BSA (NEB), 8 μM random hexamers (Promega), 8 μM oligo d(T) (Promega), 80 U RNAsin (Promega), and 500 U Superscript II reverse transcriptase (Invitrogen).

    Sonication:

    Article Title: The MIRA method for DNA methylation analysis
    Article Snippet: 1 MseI enzyme, NEB 2 buffer and 1 mg/mL BSA (New England Biolabs; Ipswich, MA). .. 1 MseI enzyme, NEB 2 buffer and 1 mg/mL BSA (New England Biolabs; Ipswich, MA).

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation. .. After proximity ligation, nuclei were pelleted by centrifugation at 2500 r cf. for 5 minutes and resuspended in 1 mL of ChIP sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS with protease inhibitor).

    Binding Assay:

    Article Title: Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae
    Article Snippet: The 341 bp Eco RI– Bam HI fragment of pMB1 ( , ) containing three UASs from the nifH – nifJ intergenic region, was 5′-end labelled using [γ-32 P]dATP and T4 polynucleotide kinase. .. Reactions for the binding assays contained 5 nM DNA, 8.1 ng/µl poly(dI–dC) (Boehringer Mannheim), 25 mM Tris acetate pH 8, 50 mM potassium acetate, 13.5 mM ammonium acetate, 0.5 µM acetylated BSA (NEB) and 2.5–7.5 µM NifA C-terminal DNA-binding domain in a final volume of 15 µl. .. The reaction mixes were incubated at 30°C for 20 min. DNase I (1.1 × 10–4 U; Boehringer Mannheim) was added in 3 µl of buffer containing 10 mM Tris–HCl, 10 mM CaCl2 , 40 mM MgCl2 and 10% glycerol.

    Hi-C:

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: Paragraph title: Hi-C, ChIA-PET, and HiChIP Library Preparation and Processing ... This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202).

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Nuclei were pelleted by centrifugation at 2500 r cf. for 5 min at 4°C and washed once with 500 μL of ice-cold Hi-C lysis buffer. .. Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation.

    Molecular Weight:

    Article Title: Reconstituting the 4-Strand DNA Strand Exchange
    Article Snippet: Standard agarose gel running setup (Bio-Rad Wide Mini-Sub Cell GT) AlphaImager 3400 gel documentation station (Alpha Innotech) Vortex-2 Genie (Scientific Industries) Low-speed benchtop centrifuge Heat blocks or water baths set at 37°C and 65°C UV lamp Electroelution apparatus: Schleicher & Schuell Elutrap (Whatman) Micro Bio-Spin 6 columns (Bio-Rad, cat# 732-6221) 50-μL quartz cuvettes (Agilent Technologies, cat# 5062-2496) .. NanoPure water Xho I restriction endonucleases (NEB) Alw NI restriction endonucleases (NEB) Bovine serum albumin (BSA), 20 mg/mL (NEB, cat# B9000S) Ethanol 200 proof TAE buffer: 40 m M Tris, 20 m M acetic acid, 1 m M EDTA, pH 8.0 TE buffer: 10 m M Tris–HCl, 1 m M EDTA, pH 8.0 Agarose, Type I-A, Low-EEO (Sigma cat# A0169) for analytical electrophoresis Agarose, Certified Molecular Biology (Bio-Rad cat# 161-3101) for purification of dsDNA and gapped DNA molecules by electroelution 1-kb DNA ladder Molecular Weight Marker 10 × DNA loading buffer for agarose gels (0.25% bromophenol blue, 50% glycerol, 1 m M EDTA, pH 8.0) Ethidium bromide staining solution, 2 μg/mL in water 1-Butanol ≥99.4% ACS reagent (Sigma cat# 360465) 10 × annealing buffer: 250 m M Tris-acetate, pH 7.5, 500 m M NaCl Formamide ≥99.5% (Sigma cat# F9037) .. Carry out the cleavage of pBS II SK (+) plasmid dsDNA with Xho I and Alw NI restriction endonucleases.

    DNA Extraction:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: Paragraph title: DNA extraction and Sanger sequencing ... BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Enzyme Activity Assay:

    Article Title: Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
    Article Snippet: Authentic coniferyl p -coumarate 3Ga and sinapyl p -coumarate 3Sa were synthesized as described previously ( ). p -Coumaryl p -coumarate 3Ha and sinapyl acetate were made by an analogous route ( ). .. The OsPMT enzyme activity assay, in 50 m m sodium phosphate buffer, pH 6, containing 1 m m dithiothreitol (DTT), 1 m m CoA thioester, 1 m m monolignol, and deionized water to produce a final volume of 50 μl, was initiated by adding of 1 μg of purified PMT protein in 1× BSA (NEB). .. After a 30-min incubation, the reaction was stopped by the addition of 100 m m hydrochloric acid.

    Magnetic Beads:

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202). .. Cell debris was pelleted and the supernatant was transferred into a new 1.5 ml tube for immunoprecipitation.

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: Taken together, these findings suggest that SauCas9 may be an attractive alternative or complement to SpyCas9 and could possibly be leveraged for future biotechnological and therapeutic applications. .. S. pyogenes Cas9 (# M0386M), EnGen sgRNA Synthesis Kit, S. pyogenes (# E3322S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S), 2× RNA Loading Dye (# B0363S), Nucleoside Digestion Mix (M0649S), Q5 Hot Start High-Fidelity 2× Master Mix (# M0494L), Monarch PCR & DNA Cleanup Kit (# T1030L), Proteinase K (# P8107S), Shrimp Alkaline Phosphatase (# M0371S), T4 Polynucleotide Kinase (# M0201S), Streptavidin Magnetic Beads (# S1420S), and NEBuffer 3.1 (# B7203S) with a 1× composition of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 at 25°C were all from New England Biolabs. .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques.

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation. .. Nuclei were sonicated using a Covaris S220 for 6 minutes with the following settings: fill level 8, duty cycle 5, peak incidence power 140, cycles per burst 200.

    Microscopy:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: Tumor and non-tumor areas were separately microdissected from sections of archival paraffin-embedded tissue using a dissection microscope. .. BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Purification:

    Article Title: Reconstituting the 4-Strand DNA Strand Exchange
    Article Snippet: Standard agarose gel running setup (Bio-Rad Wide Mini-Sub Cell GT) AlphaImager 3400 gel documentation station (Alpha Innotech) Vortex-2 Genie (Scientific Industries) Low-speed benchtop centrifuge Heat blocks or water baths set at 37°C and 65°C UV lamp Electroelution apparatus: Schleicher & Schuell Elutrap (Whatman) Micro Bio-Spin 6 columns (Bio-Rad, cat# 732-6221) 50-μL quartz cuvettes (Agilent Technologies, cat# 5062-2496) .. NanoPure water Xho I restriction endonucleases (NEB) Alw NI restriction endonucleases (NEB) Bovine serum albumin (BSA), 20 mg/mL (NEB, cat# B9000S) Ethanol 200 proof TAE buffer: 40 m M Tris, 20 m M acetic acid, 1 m M EDTA, pH 8.0 TE buffer: 10 m M Tris–HCl, 1 m M EDTA, pH 8.0 Agarose, Type I-A, Low-EEO (Sigma cat# A0169) for analytical electrophoresis Agarose, Certified Molecular Biology (Bio-Rad cat# 161-3101) for purification of dsDNA and gapped DNA molecules by electroelution 1-kb DNA ladder Molecular Weight Marker 10 × DNA loading buffer for agarose gels (0.25% bromophenol blue, 50% glycerol, 1 m M EDTA, pH 8.0) Ethidium bromide staining solution, 2 μg/mL in water 1-Butanol ≥99.4% ACS reagent (Sigma cat# 360465) 10 × annealing buffer: 250 m M Tris-acetate, pH 7.5, 500 m M NaCl Formamide ≥99.5% (Sigma cat# F9037) .. Carry out the cleavage of pBS II SK (+) plasmid dsDNA with Xho I and Alw NI restriction endonucleases.

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: DNA was extracted and purified with a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Article Title: Specific DNA-binding by Apicomplexan AP2 transcription factors
    Article Snippet: Primer extension from a universal 24-mer region on all 60-mers generates a double-stranded DNA microarray platform. .. Purified proteins were diluted to a final concentration of 100–500 nM in PBS, 2% (wt/vol) milk, 51.3 ng/μl salmon testes DNA (Sigma), 0.2 μg/μl BSA (New England Biolabs), and incubated for 1 h at 20°C. .. After washing, specific DNA–protein interactions were visualized by using a GSI Lumonics ScanArray 5000 scanner to detect fluorescence from an Alexa488-conjugated anti-GST antibody.

    Article Title: Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
    Article Snippet: Authentic coniferyl p -coumarate 3Ga and sinapyl p -coumarate 3Sa were synthesized as described previously ( ). p -Coumaryl p -coumarate 3Ha and sinapyl acetate were made by an analogous route ( ). .. The OsPMT enzyme activity assay, in 50 m m sodium phosphate buffer, pH 6, containing 1 m m dithiothreitol (DTT), 1 m m CoA thioester, 1 m m monolignol, and deionized water to produce a final volume of 50 μl, was initiated by adding of 1 μg of purified PMT protein in 1× BSA (NEB). .. After a 30-min incubation, the reaction was stopped by the addition of 100 m m hydrochloric acid.

    Sequencing:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: Paragraph title: DNA extraction and Sanger sequencing ... BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics. .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics.

    Software:

    Article Title: A distinct subset of Atypical Spitz Tumors is characterized by BRAF mutation and loss of BAP1 expression
    Article Snippet: BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer. .. BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), 1× Hotstar Taq buffer (Qiagen) and 0.4 µM primer.

    Article Title: Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
    Article Snippet: Purified CoA thioesters were analyzed for purity using an Acquity Ultra Performance LC with an Acquity UPLC BEH C18 1.7 μm 2.1 ×100-mm column and the Acquity Console and Empower 2 Software (Waters Corporation). .. The OsPMT enzyme activity assay, in 50 m m sodium phosphate buffer, pH 6, containing 1 m m dithiothreitol (DTT), 1 m m CoA thioester, 1 m m monolignol, and deionized water to produce a final volume of 50 μl, was initiated by adding of 1 μg of purified PMT protein in 1× BSA (NEB).

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics. .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics.

    Immunoprecipitation:

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202). .. Following ligation, nuclei were pelleted and resuspended in 200 µl cold Nuclei Lysis Buffer (50 mM Tris-HCl pH 9, 10 mM EDTA, 1% SDS, and 1× Protease Inhibitors) with incubation on ice for 20 min. After incubation we added 100 µl cold IP Dilution Buffer (0.01% SDS, 1.1% Trition X-100, 1.2 mM EDTA, 16.7 Tris-HCl pH 8, 16.7 mM NaCl, and 1× Protease Inhibitors) and sonicated to approximately 250 bp fragments.

    HiChIP:

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: Paragraph title: Hi-C, ChIA-PET, and HiChIP Library Preparation and Processing ... This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202).

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Paragraph title: HiChIP ... Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation.

    Lysis:

    Article Title: Editing DNA methylation in the mammalian genome
    Article Snippet: Cell pellet was re-suspended with 550 µl lysis buffer (10 mM Tris-HCl with pH 8.0, 10 mM NaCl, and 0.2% IGEPAL CA630 with proteinase inhibitor), and incubated on ice for 20 min. .. Then 713 µl H2O, 120 µl 10 x T4 DNA ligase buffer (NEB, B0202), 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl T4 DNA ligase (NEB, M0202) were added and incubated for 22 hour at 16 °C.

    Article Title: Evolutionarily Conserved Principles Predict 3D Chromatin Organization
    Article Snippet: Cells were pelleted and resuspended in 500 µl cold Hi-C lysis buffer (10 mM Tris-HCl pH8, 10 mM NaCl, 0.2% Igepal CA-630, and 1× Protease Inhibitor (Roche 11873580001) and incubated on ice for 1 h. Nuclei were pelleted at 2500 rcf for 5 min at 4°C, resuspended in 100 µl 0. .. This reaction was placed at 37°C for 1.5 h, after which samples were ligated for 4 h at room temperature with addition of 663 µl H2 O, 120 µl 10× NEB T4 DNA Ligase buffer, 100 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, and 5 µl 400 u/µl T4 DNA Ligase (NEB M0202).

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: Nuclei were pelleted by centrifugation at 2500 r cf. for 5 min at 4°C and washed once with 500 μL of ice-cold Hi-C lysis buffer. .. Proximity ligation was performed by addition of 947 μL of ligation master mix, containing 150 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 μL of 10% Triton X-100, 7.5 μL of 20 mg/mL BSA (NEB, B9000), 10 μL of 400 U/μL T4 DNA ligase (NEB, M0202), and 655.5 μL of water, and incubation at room temperature for 4 hours with rotation.

    IA:

    Article Title: Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin-like small archaeal modifier proteins (SAMPs) from protein-conjugates
    Article Snippet: Molecular biology grade enzymes were purchased from New England Biolabs (Ipswich, MA) unless otherwise indicated. .. Molecular biology grade enzymes were purchased from New England Biolabs (Ipswich, MA) unless otherwise indicated.

    Silver Staining:

    Article Title: Specific DNA-binding by Apicomplexan AP2 transcription factors
    Article Snippet: Purity was verified by silver stain SDS/PAGE and Western blot analysis with an anti-GST antibody (Invitrogen). .. Purified proteins were diluted to a final concentration of 100–500 nM in PBS, 2% (wt/vol) milk, 51.3 ng/μl salmon testes DNA (Sigma), 0.2 μg/μl BSA (New England Biolabs), and incubated for 1 h at 20°C.

    Liquid Chromatography:

    Article Title: Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
    Article Snippet: Purified CoA thioesters were analyzed for purity using an Acquity Ultra Performance LC with an Acquity UPLC BEH C18 1.7 μm 2.1 ×100-mm column and the Acquity Console and Empower 2 Software (Waters Corporation). .. The OsPMT enzyme activity assay, in 50 m m sodium phosphate buffer, pH 6, containing 1 m m dithiothreitol (DTT), 1 m m CoA thioester, 1 m m monolignol, and deionized water to produce a final volume of 50 μl, was initiated by adding of 1 μg of purified PMT protein in 1× BSA (NEB).

    Plasmid Preparation:

    Article Title: A solenoid design for assessing determinants of parallel β-sheet registration
    Article Snippet: Molecular biology reagents were purchased from New England Biolabs. .. Molecular biology reagents were purchased from New England Biolabs.

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: Cosmid and plasmid DNAs were extracted from E. coli by the alkaline method ( ). .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics.

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: Ampicillin was included at 50 μg/ml when plasmid-containing E. coli strains were being grown ( ); when Streptomyces strains containing resistance-encoding plasmids were being grown, thiostrepton was included at a concentration of 50 or 5 μg/ml in solid or liquid media, respectively, and hygromycin was included at a concentration of 200 or 20 μg/ml in solid or liquid media, respectively. .. Standard molecular biology protocols ( ) and enzymes purchased from New England BioLabs or Invitrogen were used to construct various pGSP and pSCON plasmids listed in Table .

    TaqMan Assay:

    Article Title: Association between two ?-opioid receptor gene (OPRM1) haplotype blocks and drug or alcohol dependence
    Article Snippet: All other SNPs were genotyped using the TaqMan assay in a 384-well microplate format. .. Briefly, 1 ng of DNA was amplified in a final volume of 2 μl containing 0.05 μl of 20× (or 0.025 μl of 40×) MGB probes and primers, 1 μl of 2× TaqMan Universal PCR Master Mix, and 0.004 μl of 100× BSA (New England Biolabs Inc., Beverly, MA).

    Electrophoresis:

    Article Title: Reconstituting the 4-Strand DNA Strand Exchange
    Article Snippet: Standard agarose gel running setup (Bio-Rad Wide Mini-Sub Cell GT) AlphaImager 3400 gel documentation station (Alpha Innotech) Vortex-2 Genie (Scientific Industries) Low-speed benchtop centrifuge Heat blocks or water baths set at 37°C and 65°C UV lamp Electroelution apparatus: Schleicher & Schuell Elutrap (Whatman) Micro Bio-Spin 6 columns (Bio-Rad, cat# 732-6221) 50-μL quartz cuvettes (Agilent Technologies, cat# 5062-2496) .. NanoPure water Xho I restriction endonucleases (NEB) Alw NI restriction endonucleases (NEB) Bovine serum albumin (BSA), 20 mg/mL (NEB, cat# B9000S) Ethanol 200 proof TAE buffer: 40 m M Tris, 20 m M acetic acid, 1 m M EDTA, pH 8.0 TE buffer: 10 m M Tris–HCl, 1 m M EDTA, pH 8.0 Agarose, Type I-A, Low-EEO (Sigma cat# A0169) for analytical electrophoresis Agarose, Certified Molecular Biology (Bio-Rad cat# 161-3101) for purification of dsDNA and gapped DNA molecules by electroelution 1-kb DNA ladder Molecular Weight Marker 10 × DNA loading buffer for agarose gels (0.25% bromophenol blue, 50% glycerol, 1 m M EDTA, pH 8.0) Ethidium bromide staining solution, 2 μg/mL in water 1-Butanol ≥99.4% ACS reagent (Sigma cat# 360465) 10 × annealing buffer: 250 m M Tris-acetate, pH 7.5, 500 m M NaCl Formamide ≥99.5% (Sigma cat# F9037) .. Carry out the cleavage of pBS II SK (+) plasmid dsDNA with Xho I and Alw NI restriction endonucleases.

    Article Title: Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases
    Article Snippet: To provide novel insights into the mechanism of this important and unique enzyme, with potential focus on the role of the metal co-factor, we decided to investigate the contribution of Motif I aspartate to structure and function. .. Materials for electrophoresis or chromatography were from Bio-Rad or Amersham Biosciences, phenol red from Merck, molecular biology products were from New England BioLabs or Fermentas. .. Other materials were from Sigma or Stratagene.

    Recombinant:

    Article Title: Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches
    Article Snippet: T4 ligase, Bsa I, and 100 × bovine serum albumin (BSA) were purchased from NEB, USA. .. T4 ligase, Bsa I, and 100 × bovine serum albumin (BSA) were purchased from NEB, USA.

    In Situ:

    Article Title: Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes
    Article Snippet: In situ reverse transcription was performed using BNA primers positioned within ≤1 nt from the 5′ end of the padlock probe target site (see Table for nucleotide sequences). .. BNA primer hybridization and mRNA reverse transcription were performed o/n at 42 °C using 200 nM of BNA primer (IDT, Leuven Belgium), 20 U/µl RevertAid H minus M-MuLV reverse transcriptase in 1x M-MuLV reaction buffer (ThermoFisher Scientific, Waltham, MA, USA), 1 U/µl RiboLock RNase Inhibitor (ThermoFisher Scientific, Waltham, MA, USA), 500 µM dNTP and 0.2 µg/µl BSA (New England Biolabs, Ipswich, MA, USA).

    Ethanol Precipitation:

    Article Title: Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae
    Article Snippet: Reactions for the binding assays contained 5 nM DNA, 8.1 ng/µl poly(dI–dC) (Boehringer Mannheim), 25 mM Tris acetate pH 8, 50 mM potassium acetate, 13.5 mM ammonium acetate, 0.5 µM acetylated BSA (NEB) and 2.5–7.5 µM NifA C-terminal DNA-binding domain in a final volume of 15 µl. .. Reactions for the binding assays contained 5 nM DNA, 8.1 ng/µl poly(dI–dC) (Boehringer Mannheim), 25 mM Tris acetate pH 8, 50 mM potassium acetate, 13.5 mM ammonium acetate, 0.5 µM acetylated BSA (NEB) and 2.5–7.5 µM NifA C-terminal DNA-binding domain in a final volume of 15 µl.

    Concentration Assay:

    Article Title: Specific DNA-binding by Apicomplexan AP2 transcription factors
    Article Snippet: Primer extension from a universal 24-mer region on all 60-mers generates a double-stranded DNA microarray platform. .. Purified proteins were diluted to a final concentration of 100–500 nM in PBS, 2% (wt/vol) milk, 51.3 ng/μl salmon testes DNA (Sigma), 0.2 μg/μl BSA (New England Biolabs), and incubated for 1 h at 20°C. .. After washing, specific DNA–protein interactions were visualized by using a GSI Lumonics ScanArray 5000 scanner to detect fluorescence from an Alexa488-conjugated anti-GST antibody.

    Article Title: Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
    Article Snippet: The concentration for each CoA thioester was calculated based on its absorbance maximum and extinction coefficient ( , ). .. The OsPMT enzyme activity assay, in 50 m m sodium phosphate buffer, pH 6, containing 1 m m dithiothreitol (DTT), 1 m m CoA thioester, 1 m m monolignol, and deionized water to produce a final volume of 50 μl, was initiated by adding of 1 μg of purified PMT protein in 1× BSA (NEB).

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: S. pyogenes Cas9 (# M0386M), EnGen sgRNA Synthesis Kit, S. pyogenes (# E3322S), HiScribe T7 High Yield RNA Synthesis Kit (E2040S), 2× RNA Loading Dye (# B0363S), Nucleoside Digestion Mix (M0649S), Q5 Hot Start High-Fidelity 2× Master Mix (# M0494L), Monarch PCR & DNA Cleanup Kit (# T1030L), Proteinase K (# P8107S), Shrimp Alkaline Phosphatase (# M0371S), T4 Polynucleotide Kinase (# M0201S), Streptavidin Magnetic Beads (# S1420S), and NEBuffer 3.1 (# B7203S) with a 1× composition of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 at 25°C were all from New England Biolabs. .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques.

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: Ampicillin was included at 50 μg/ml when plasmid-containing E. coli strains were being grown ( ); when Streptomyces strains containing resistance-encoding plasmids were being grown, thiostrepton was included at a concentration of 50 or 5 μg/ml in solid or liquid media, respectively, and hygromycin was included at a concentration of 200 or 20 μg/ml in solid or liquid media, respectively. .. Standard molecular biology protocols ( ) and enzymes purchased from New England BioLabs or Invitrogen were used to construct various pGSP and pSCON plasmids listed in Table .

    Pulsed-Field Gel:

    Article Title: Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors in Streptomyces ambofaciens
    Article Snippet: Standard or pulsed-field gel electrophoresis DNA preparations from S. ambofaciens were made as described earlier ( , ). .. All restriction enzymes, PCR reagents, and other molecular biology reagents were purchased from New England Biolabs or Roche Diagnostics.

    Marker:

    Article Title: Reconstituting the 4-Strand DNA Strand Exchange
    Article Snippet: Standard agarose gel running setup (Bio-Rad Wide Mini-Sub Cell GT) AlphaImager 3400 gel documentation station (Alpha Innotech) Vortex-2 Genie (Scientific Industries) Low-speed benchtop centrifuge Heat blocks or water baths set at 37°C and 65°C UV lamp Electroelution apparatus: Schleicher & Schuell Elutrap (Whatman) Micro Bio-Spin 6 columns (Bio-Rad, cat# 732-6221) 50-μL quartz cuvettes (Agilent Technologies, cat# 5062-2496) .. NanoPure water Xho I restriction endonucleases (NEB) Alw NI restriction endonucleases (NEB) Bovine serum albumin (BSA), 20 mg/mL (NEB, cat# B9000S) Ethanol 200 proof TAE buffer: 40 m M Tris, 20 m M acetic acid, 1 m M EDTA, pH 8.0 TE buffer: 10 m M Tris–HCl, 1 m M EDTA, pH 8.0 Agarose, Type I-A, Low-EEO (Sigma cat# A0169) for analytical electrophoresis Agarose, Certified Molecular Biology (Bio-Rad cat# 161-3101) for purification of dsDNA and gapped DNA molecules by electroelution 1-kb DNA ladder Molecular Weight Marker 10 × DNA loading buffer for agarose gels (0.25% bromophenol blue, 50% glycerol, 1 m M EDTA, pH 8.0) Ethidium bromide staining solution, 2 μg/mL in water 1-Butanol ≥99.4% ACS reagent (Sigma cat# 360465) 10 × annealing buffer: 250 m M Tris-acetate, pH 7.5, 500 m M NaCl Formamide ≥99.5% (Sigma cat# F9037) .. Carry out the cleavage of pBS II SK (+) plasmid dsDNA with Xho I and Alw NI restriction endonucleases.

    Staining:

    Article Title: Reconstituting the 4-Strand DNA Strand Exchange
    Article Snippet: Standard agarose gel running setup (Bio-Rad Wide Mini-Sub Cell GT) AlphaImager 3400 gel documentation station (Alpha Innotech) Vortex-2 Genie (Scientific Industries) Low-speed benchtop centrifuge Heat blocks or water baths set at 37°C and 65°C UV lamp Electroelution apparatus: Schleicher & Schuell Elutrap (Whatman) Micro Bio-Spin 6 columns (Bio-Rad, cat# 732-6221) 50-μL quartz cuvettes (Agilent Technologies, cat# 5062-2496) .. NanoPure water Xho I restriction endonucleases (NEB) Alw NI restriction endonucleases (NEB) Bovine serum albumin (BSA), 20 mg/mL (NEB, cat# B9000S) Ethanol 200 proof TAE buffer: 40 m M Tris, 20 m M acetic acid, 1 m M EDTA, pH 8.0 TE buffer: 10 m M Tris–HCl, 1 m M EDTA, pH 8.0 Agarose, Type I-A, Low-EEO (Sigma cat# A0169) for analytical electrophoresis Agarose, Certified Molecular Biology (Bio-Rad cat# 161-3101) for purification of dsDNA and gapped DNA molecules by electroelution 1-kb DNA ladder Molecular Weight Marker 10 × DNA loading buffer for agarose gels (0.25% bromophenol blue, 50% glycerol, 1 m M EDTA, pH 8.0) Ethidium bromide staining solution, 2 μg/mL in water 1-Butanol ≥99.4% ACS reagent (Sigma cat# 360465) 10 × annealing buffer: 250 m M Tris-acetate, pH 7.5, 500 m M NaCl Formamide ≥99.5% (Sigma cat# F9037) .. Carry out the cleavage of pBS II SK (+) plasmid dsDNA with Xho I and Alw NI restriction endonucleases.

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    New England Biolabs bsa i bsa i hf
    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease <t>Bsa</t> I and T4 DNA ligase. The overhangs produced by Bsa I are colored
    Bsa I Bsa I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa i bsa i hf/product/New England Biolabs
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsa i bsa i hf - by Bioz Stars, 2019-12
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    81
    New England Biolabs restriction enzyme bsa xi
    M13 PCR products (10 μl) of <t>JAK2</t> clones were loaded onto a 6% PAGE to allow visualization of the expected 266-bp amplicon according to Materials and Methods. Afterward, 10 μl of PCR product of each clone was digested with <t>Bsa</t> XI for 16
    Restriction Enzyme Bsa Xi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 81/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme bsa xi/product/New England Biolabs
    Average 81 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme bsa xi - by Bioz Stars, 2019-12
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    99
    New England Biolabs bsa i hf
    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease <t>Bsa</t> I and T4 DNA ligase. The overhangs produced by Bsa I are colored
    Bsa I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa i hf/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    bsa i hf - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    Image Search Results


    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Article Snippet: Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Article Snippet: Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Plasmid Preparation, Expressing, Construct

    Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Article Snippet: Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).

    Techniques: Clone Assay, Expressing, Plasmid Preparation

    M13 PCR products (10 μl) of JAK2 clones were loaded onto a 6% PAGE to allow visualization of the expected 266-bp amplicon according to Materials and Methods. Afterward, 10 μl of PCR product of each clone was digested with Bsa XI for 16

    Journal:

    Article Title: Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

    doi: 10.2353/jmoldx.2006.050064

    Figure Lengend Snippet: M13 PCR products (10 μl) of JAK2 clones were loaded onto a 6% PAGE to allow visualization of the expected 266-bp amplicon according to Materials and Methods. Afterward, 10 μl of PCR product of each clone was digested with Bsa XI for 16

    Article Snippet: Notably, the JAK2 mutation destroys a recognition site for the restriction enzyme Bsa XI, leaving mutated PCR products undigested, whereas unmutated products showed complete digestion.

    Techniques: Polymerase Chain Reaction, Clone Assay, Polyacrylamide Gel Electrophoresis, Amplification

    Identical sequence segment as shown in marked the recognition site for Bsa XI. Relevant nucleotides are in bold . A: Triangles mark the two cut sites of Bsa XI leading to digestion of the amplified JAK2 (entire amplicon size italicized) in fragments

    Journal:

    Article Title: Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

    doi: 10.2353/jmoldx.2006.050064

    Figure Lengend Snippet: Identical sequence segment as shown in marked the recognition site for Bsa XI. Relevant nucleotides are in bold . A: Triangles mark the two cut sites of Bsa XI leading to digestion of the amplified JAK2 (entire amplicon size italicized) in fragments

    Article Snippet: Notably, the JAK2 mutation destroys a recognition site for the restriction enzyme Bsa XI, leaving mutated PCR products undigested, whereas unmutated products showed complete digestion.

    Techniques: Sequencing, Amplification

    Six-percent PAGE was loaded with Bsa XI-digested JAK2 products derived from the granulocyte fraction and/or the mononuclear cell fraction of peripheral blood and/or bone marrow aspirates of four patients (1 to 3 with a history of PV; 4 and 5 with suspected

    Journal:

    Article Title: Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

    doi: 10.2353/jmoldx.2006.050064

    Figure Lengend Snippet: Six-percent PAGE was loaded with Bsa XI-digested JAK2 products derived from the granulocyte fraction and/or the mononuclear cell fraction of peripheral blood and/or bone marrow aspirates of four patients (1 to 3 with a history of PV; 4 and 5 with suspected

    Article Snippet: Notably, the JAK2 mutation destroys a recognition site for the restriction enzyme Bsa XI, leaving mutated PCR products undigested, whereas unmutated products showed complete digestion.

    Techniques: Polyacrylamide Gel Electrophoresis, Derivative Assay

    A: JAK2 PCR products derived from a PV patient carrying the JAK2 mutation ( 1a – 1d ) and a control sample ( 2a – 2d ) were digested with 4 U of Bsa XI for 2, 4, 6, and 16 hours. B: In the control sample, virtually complete digestion of the initial

    Journal:

    Article Title: Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

    doi: 10.2353/jmoldx.2006.050064

    Figure Lengend Snippet: A: JAK2 PCR products derived from a PV patient carrying the JAK2 mutation ( 1a – 1d ) and a control sample ( 2a – 2d ) were digested with 4 U of Bsa XI for 2, 4, 6, and 16 hours. B: In the control sample, virtually complete digestion of the initial

    Article Snippet: Notably, the JAK2 mutation destroys a recognition site for the restriction enzyme Bsa XI, leaving mutated PCR products undigested, whereas unmutated products showed complete digestion.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Mutagenesis

    A representative 6% PAGE loaded with JAK2 products after digestion with Bsa XI according to Materials and Methods. Lanes 1 to 10 show mutated ( 2 , 4 , 5 , 6 , and 10 ) as well as unmutated ( 1 , 3 , 7 , 8 , and 9 ) cases with advanced cIMF together with a positive

    Journal:

    Article Title: Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

    doi: 10.2353/jmoldx.2006.050064

    Figure Lengend Snippet: A representative 6% PAGE loaded with JAK2 products after digestion with Bsa XI according to Materials and Methods. Lanes 1 to 10 show mutated ( 2 , 4 , 5 , 6 , and 10 ) as well as unmutated ( 1 , 3 , 7 , 8 , and 9 ) cases with advanced cIMF together with a positive

    Article Snippet: Notably, the JAK2 mutation destroys a recognition site for the restriction enzyme Bsa XI, leaving mutated PCR products undigested, whereas unmutated products showed complete digestion.

    Techniques: Polyacrylamide Gel Electrophoresis

    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Plasmid Preparation, Expressing, Construct

    Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Journal: Microbial Cell Factories

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    doi: 10.1186/s12934-017-0637-5

    Figure Lengend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown

    Article Snippet: The better performance of the wild-type enzyme may be caused by its full activity at 50 °C during the final restriction step, in contrast to the depleted activity of Bsa I-HF (NEB, personal communication).

    Techniques: Clone Assay, Expressing, Plasmid Preparation

    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Article Snippet: We found both Bsa I-HF from NewEngland Biolabs, as well as the isoschizomer FastDigest Eco 31I from Fermentas to be highly sensitive to temperature fluctuations in a batch dependent manner.

    Techniques: Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Sequencing, Selection, Transgenic Assay

    Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Article Snippet: We found both Bsa I-HF from NewEngland Biolabs, as well as the isoschizomer FastDigest Eco 31I from Fermentas to be highly sensitive to temperature fluctuations in a batch dependent manner.

    Techniques: Expressing, Plasmid Preparation, Sequencing, Hemagglutination Assay, Fluorescence, Microscopy, Construct, Methylation, Transformation Assay, Isolation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Article Snippet: We found both Bsa I-HF from NewEngland Biolabs, as well as the isoschizomer FastDigest Eco 31I from Fermentas to be highly sensitive to temperature fluctuations in a batch dependent manner.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated