atp  (New England Biolabs)


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    Name:
    Adenosine 5 Triphosphate ATP
    Description:
    Adenosine 5 Triphosphate ATP 5 ml
    Catalog Number:
    p0756l
    Price:
    140
    Size:
    5 ml
    Category:
    Nucleotides
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    Structured Review

    New England Biolabs atp
    Adenosine 5 Triphosphate ATP
    Adenosine 5 Triphosphate ATP 5 ml
    https://www.bioz.com/result/atp/product/New England Biolabs
    Average 95 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling"

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    Journal: mBio

    doi: 10.1128/mBio.00833-16

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Figure Legend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Techniques Used: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.
    Figure Legend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Techniques Used: Western Blot

    2) Product Images from "Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579"

    Article Title: Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.06248-11

    Characterization of BceSIV endonuclease. (A) Schematic diagram of six ORFs of the BceSIV operon. The gene identifier (Bce_0364 to Bce_0370) is indicated below the ORFs. Bce_0364, a possible transcription regulator, similar to phage repressors; Bce_0365, M.BceSIV (GCWGC); Bce_0367, unknown function, no apparent endonuclease activity; Bce_0368, R1 subunit of BceSIV, NTPase/helicase; Bce_0369, BceSIV endonuclease catalytic subunit, R2 subunit; Bce_0370, putative transcription regular, similar to the controller (C) protein of other R-M systems. (B) Digestion of pBR322 DNA by cell extracts containing BceSIV R1, R2, and R1+R2 in the presence or absence of ATP (2 mM). Lanes 1 and 13, 1-kb DNA ladder; lanes 2 to 11 contain either R1, R2, or R1+R2 as indicated; lane 12, linear DNA; lane 14, nicked circular DNA; lane 15, uncut pBR322 DNA. (C) Purified BceSIV R1 subunit from a chitin column. The predicted molecular mass of R1 is 69.6 kDa. Lanes 1 to 9, eluted fractions containing R1; lane 10, total protein from the chitin column (R1-intein-CBD fusion protein, R1, and intein-CBD). (D) Purified BceSIV R1 and R2 subunits from a chitin column (lanes 1 to 9). The predicted molecular mass of R2 is 42.7 kDa. Lane 10, total protein from the chitin column. (E) BceSIV digestion of pBR322 in the presence of GTP, γ-S-GTP (nonhydrolyzable GTP analog), ATP, and UTP. (F) BceSIV digestion of pBR322 in the presence of ATP in the range of 0.4 mM to 20 mM. M, 1 kb DNA ladder. (G) BceSIV digestion of pBR322 in the presence of GTP in the range of 0.4 mM to 20 mM. Lanes M, 1-kb DNA ladder.
    Figure Legend Snippet: Characterization of BceSIV endonuclease. (A) Schematic diagram of six ORFs of the BceSIV operon. The gene identifier (Bce_0364 to Bce_0370) is indicated below the ORFs. Bce_0364, a possible transcription regulator, similar to phage repressors; Bce_0365, M.BceSIV (GCWGC); Bce_0367, unknown function, no apparent endonuclease activity; Bce_0368, R1 subunit of BceSIV, NTPase/helicase; Bce_0369, BceSIV endonuclease catalytic subunit, R2 subunit; Bce_0370, putative transcription regular, similar to the controller (C) protein of other R-M systems. (B) Digestion of pBR322 DNA by cell extracts containing BceSIV R1, R2, and R1+R2 in the presence or absence of ATP (2 mM). Lanes 1 and 13, 1-kb DNA ladder; lanes 2 to 11 contain either R1, R2, or R1+R2 as indicated; lane 12, linear DNA; lane 14, nicked circular DNA; lane 15, uncut pBR322 DNA. (C) Purified BceSIV R1 subunit from a chitin column. The predicted molecular mass of R1 is 69.6 kDa. Lanes 1 to 9, eluted fractions containing R1; lane 10, total protein from the chitin column (R1-intein-CBD fusion protein, R1, and intein-CBD). (D) Purified BceSIV R1 and R2 subunits from a chitin column (lanes 1 to 9). The predicted molecular mass of R2 is 42.7 kDa. Lane 10, total protein from the chitin column. (E) BceSIV digestion of pBR322 in the presence of GTP, γ-S-GTP (nonhydrolyzable GTP analog), ATP, and UTP. (F) BceSIV digestion of pBR322 in the presence of ATP in the range of 0.4 mM to 20 mM. M, 1 kb DNA ladder. (G) BceSIV digestion of pBR322 in the presence of GTP in the range of 0.4 mM to 20 mM. Lanes M, 1-kb DNA ladder.

    Techniques Used: Activity Assay, Purification

    3) Product Images from "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)"

    Article Title: Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/915061

    Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.
    Figure Legend Snippet: Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.

    Techniques Used: Ligation, Clone Assay, Blocking Assay

    4) Product Images from "Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival"

    Article Title: Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival

    Journal: Nature cell biology

    doi: 10.1038/ncb3194

    Proposed working-model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival
    Figure Legend Snippet: Proposed working-model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival

    Techniques Used:

    Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch
    Figure Legend Snippet: Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch

    Techniques Used:

    PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells
    Figure Legend Snippet: PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells

    Techniques Used:

    5) Product Images from "The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma"

    Article Title: The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00578-5

    FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram
    Figure Legend Snippet: FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram

    Techniques Used: In Vitro, In Vivo, Recombinant, Mutagenesis, Activity Assay, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, SDS Page, Purification, Incubation, Stable Transfection, Expressing, Immunoprecipitation, Staining

    6) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    Related Articles

    Diagnostic Assay:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Amplification:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Library preparation and sequencing for amplicons The detailed protocol of target deep DNA sequencing was as follows: A. Multiplex PCR amplification: The 128 amplicons were amplified by multiplex PCR on Veriti 96-well Thermal Cycler (Applied Biosystems, Waltham, MA, USA), which was performed using 30 ng genomic DNA, 15 μL Primer mix/pool (2 pools in total), 10 μL Q5 reaction buffer (NEB, Ipswich, MA, USA, Cat. B9027S), 10 μL Q5 high GC enhancer (NEB, Cat. B9028A), 1.5 μL dNTPs mix (NEB, Cat. N0447S), 0.5 μL Q5 high-fidelity DNA polymerase (NEB, Cat. M0491L), and ddH2 O to make the final reaction volume to 50 μL. .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Synthesized:

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs). .. The phosphorylated oligonucleotides were annealed to single-strand template DNA (20 μg) containing the wild-type ubiquitin sequence fused with the M13 major coat protein P3 [described by McLaughlin and Sidhu ( )] by incubating at 90°C for 3 min, at 50°C for 3 min, and at 20°C for 5 min. Complementary DNA primed by the phospho-oligonucleotides was synthesized and ligated by the addition of 10 μl of 10 mM ATP, 10 μl of a 10 mM deoxynucleotide triphosphate mixture, 15 μl of 100 mM DTT, 30 Weiss units of T4 DNA ligase, and 30 U of T7 DNA at 20°C overnight.

    Article Title: Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia
    Article Snippet: ATP (P0756S), apyrase (M0393S), trypsin (P8101S), GluC (P8100S) and the restriction endonucleases were purchased from New England Biolabs. .. S-nitrosoglutathione (GSNO) and S-nitrosocysteine (SNOC) were synthesized from glutathione using acidified nitrite.

    Construct:

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: Phage display screening The ubiquitin variant library was constructed using oligonucleotide-directed mutagenesis ( ). .. The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs).

    Incubation:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler. .. D. Column purification: The PCR products were purified with Universal DNA Purification Kit (TIANGEN, Cat. DP214-03).

    Article Title: Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
    Article Snippet: .. Substrates and enzymes were incubated in Ub-AMC assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mg/ml ovalbumin [RPI Corp, 9006-59-1], 1 mM EDTA, 5 mM MgCl2 , 1 mM ATP [New England BioLabs, P0756L]). .. After adding of Ub-AMC, the reaction was initiated, and an Envision plate reader (PerkinElmer, MA, USA) was used to measure mixture at Ex345/Em445.

    Article Title: Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia
    Article Snippet: Rat hippocampal tissues were homogenized in lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP40, 1 mM DTT, 2 mM ATP [New England Biolabs, P0756S]). .. Lysate (250 µl) containing 3 μg of protein were incubated with 40 nM aminomethylcoumarin (AMC)-linked synthetic peptide substrates for 30 min at 37°C.

    Article Title: IDH3α regulates one-carbon metabolism in glioblastoma
    Article Snippet: The product band was excised, extracted, and treated with 10 U of T4 PNK (catalog no. M0201, New England Biolabs) in 1× of T4 PNK buffer (catalog no. B0201, New England Biolabs), 1 mM ATP (catalog no. P0756, New England Biolabs), and nuclease-free water (total volume, 50 μl). .. The samples were then incubated at 37°C for 30 min and heat-inactivated at 65°C for 20 min. One microliter of T4 DNA ligase (catalog no. M1804, Promega) was added to the reaction tube.

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples
    Article Snippet: After incubating the plate at 37 °C for 30 min, we used I-DOT again to dispense 300 nl per well of CUTseq adapter at 33 nM, using a differently barcoded adapter for each well, followed by 200 nl of T4 rapid DNA ligase (Thermo Fisher Scientific, catalog number K1423), 300 nl of T4 ligase buffer 5× (Thermo Fisher Scientific, catalog number K1423), 120 nl of ATP at 10 μΜ (NEB, catalog number P0756L), 30 nl of 50 mg/ml bovine serum albumin (Thermo Fisher Scientific, catalog number AM2616), and 50 nl of nuclease-free water (Thermo Fisher Scientific, catalog number 4387936). .. We incubated the plate at 25 °C for 30 min and then pooled all the contents of the 96 wells used into 1 tube, before proceeding to IVT and library preparation following the standard CUTseq protocol.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. ..

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Immunoprecipitation used either protein G–Sepharose or GSH beads, and these were then washed three times with kinase wash buffer (25 m m Tris-HCl, pH 7.5, 70 m m NaCl, 10 m m MgCl2 , 1 m m DTT) and resuspended in kinase reaction buffer (kinase wash buffer, 10 μ m ATP (New England Biolabs, P0756), [γ-32 P]ATP for phosphorylation of full-length SF3B1 only (5 μCi/reaction; 1 Ci = 37 GBq), 0.5 μg of active cyclin E-CDK2 (Millipore, 14-475) followed by incubation at 37 °C. .. Synchronized HeLa cells were resuspended in TRIzol (Thermo Fisher Scientific, 15596026).

    Activity Assay:

    Article Title: Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia
    Article Snippet: Paragraph title: Proteasome activity assay ... Rat hippocampal tissues were homogenized in lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP40, 1 mM DTT, 2 mM ATP [New England Biolabs, P0756S]).

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: T7 ligase has 1000-fold higher activity on sticky ends than blunt ends and higher overall activity than commercially available concentrated T4 ligases. .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354)

    Mass Spectrometry:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. ..

    Kinase Assay:

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. ..

    Transformation Assay:

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs). .. The double-stranded DNA was purified using a QIAquick DNA purification kit and transformed into Escherichia coli SS320 cells preinfected with M13KO7 helper phage.

    High Performance Liquid Chromatography:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. .. Tryptic peptides were loaded onto a HPLC Chip (160 nl high-capacity sample enrichment column and 75 μ m × 150 mm SB-C18 separation column; Agilent Technologies, Santa Clara, CA, USA) and separated by flow rate at 300 nl per minute, with solvent A (0.2% (v/v) formic acid in water) and solvent B (100% acetonitrile) and the following gradients: at 0, 50, 54 and 56 min after injection with 3, 35, 80 and 100% solvent B, respectively.

    Flow Cytometry:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. .. Tryptic peptides were loaded onto a HPLC Chip (160 nl high-capacity sample enrichment column and 75 μ m × 150 mm SB-C18 separation column; Agilent Technologies, Santa Clara, CA, USA) and separated by flow rate at 300 nl per minute, with solvent A (0.2% (v/v) formic acid in water) and solvent B (100% acetonitrile) and the following gradients: at 0, 50, 54 and 56 min after injection with 3, 35, 80 and 100% solvent B, respectively.

    Ligation:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler. .. E. Adapter ligation: The mixture of 25 μL A-tailed DNA, 1 μL of 50 μmol/L multiplexing adapter, 3 μL of 10× T4 DNA ligase buffer (NEB, Cat. B0202), and 1 μL of 400 units/μL T4 DNA ligase (NEB, Cat. M0202L) was used in adapter ligation step followed by incubation at 16 °C overnight.

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples
    Article Snippet: High-throughput CUTseq To streamline the preparation of multiplexed libraries from low-input samples, we adapted the CUTseq workflow to perform the digestion and ligation steps in multi-well plates using a low-volume non-contact liquid-dispensing system (I-DOT One MC from Dispendix GmbH, Germany). .. After incubating the plate at 37 °C for 30 min, we used I-DOT again to dispense 300 nl per well of CUTseq adapter at 33 nM, using a differently barcoded adapter for each well, followed by 200 nl of T4 rapid DNA ligase (Thermo Fisher Scientific, catalog number K1423), 300 nl of T4 ligase buffer 5× (Thermo Fisher Scientific, catalog number K1423), 120 nl of ATP at 10 μΜ (NEB, catalog number P0756L), 30 nl of 50 mg/ml bovine serum albumin (Thermo Fisher Scientific, catalog number AM2616), and 50 nl of nuclease-free water (Thermo Fisher Scientific, catalog number 4387936).

    Article Title: Comprehensive whole DNA methylome analysis by integrating MeDIP-seq and MRE-seq
    Article Snippet: Denosine 5´-Triphosphate (ATP) (NEB; cat. no. P0756S). .. Reagents for adapter ligation.

    Protease Inhibitor:

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Transferring:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: Eppendorf tubes (1.5 ml) Pipette tips (10 μl, 100 μl, 1,000 μl) Deoxyguanosine triphosphate (dGTP) (New England Biolabs, catalog number: N0447S) 8-oxo-2′-deoxyguanosine-5′-Triphosphate (8-oxo-dGTP) (Jena Bioscience, catalog number: NU-1117L) DNA substrate: The DNA substrate includes a fluorescent tag at both the 5′- and 3′-ends of one of the gap-containing strand in the double-stranded DNA with a single nucleotide gap opposite template base Cytosine ( ) Note: The sequence information for the DNA substrate is presented in the Notes section of the protocol. .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes)

    DNA Sequencing:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Library preparation and sequencing for amplicons The detailed protocol of target deep DNA sequencing was as follows: A. Multiplex PCR amplification: The 128 amplicons were amplified by multiplex PCR on Veriti 96-well Thermal Cycler (Applied Biosystems, Waltham, MA, USA), which was performed using 30 ng genomic DNA, 15 μL Primer mix/pool (2 pools in total), 10 μL Q5 reaction buffer (NEB, Ipswich, MA, USA, Cat. B9027S), 10 μL Q5 high GC enhancer (NEB, Cat. B9028A), 1.5 μL dNTPs mix (NEB, Cat. N0447S), 0.5 μL Q5 high-fidelity DNA polymerase (NEB, Cat. M0491L), and ddH2 O to make the final reaction volume to 50 μL. .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Polymerase Chain Reaction:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Column purification of PCR product: All PCR products were purified using DNA Purification Kit (TIANGEN, Beijing, China, Cat. DP214-03). .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Article Title: Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome
    Article Snippet: .. Reagents Molecular biology grade water (Corning, cat. no. 46–000-CM) Oligonucleotides and primers (IDT), see Table 25 mM MgCl2 (Life Technologies, cat. no. R0971) 10 mM Tris-EDTA (TE) buffer, pH 8.0 (Quality Biological, cat. no. 351–011-131) 1 M Tris-HCl buffer, pH 8.0 (Quality Biological, cat. no. 351–007-101) Ethanol, Absolute (200 Proof), Molecular Biology Grade (Fisher Scientific, cat. no. BP2818500) (CAUTION Ethanol is highly flammable) Ase I (NEB, cat. no. R0526S) BspH I (NEB, cat. no. R0517S) BstY I (NEB, cat. no. R0523S) Hind III (NEB, cat. no. R0104S) Nco I (NEB, cat. no. R0193S) Pst I (NEB, cat. no. R0140S) RNase cocktail enzyme mix (Life Technologies, cat. no. AM2286) T4 DNA ligase (NEB, cat. no. M0202S) Adenosine 5′-Triphosphate, ATP (NEB, cat. no. P0756S) TaKaRa Ex Taq DNA polymerase, Hot-Start (Clontech, cat. no. RR006A) QiaQuick PCR Purification Kit (Qiagen, cat. no. 28106) Zymoclean Gel DNA Recovery Kit (Zymo Research, cat. no D4002) Ultrapure Agarose (Life Technologies, cat. no. 16500–100) Gel Loading Dye, 6x (NEB, cat. no. B7022S) UltraPure Tris-Acetate-EDTA (TAE) buffer, 10x (Life Technologies, cat. no. 15558–026) Ethidium Bromide solution, 10 mg/mL (Bio-Rad, cat. no. 161–0433) (CAUTION Ethidium bromide is toxic and is a potential mutagen and carcinogen.) .. 2-log ladder (NEB, cat. no. N3200S) Qubit dsDNA HS assay kit (ThermoFisher Scientific, cat. no. Q32851) Agilent DNA 1000 kit (Agilent, cat. no. 5067–1504) Agencourt AMPure XP Magnetic Beads (Beckman Coulter, cat. no. A63882) KAPA HTP Library Preparation Kit for Illumina (KAPA Biosystems, cat. no. KK8234).

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27106) QIAquick 96 PCR Purification (Qiagen, cat. no. 28181) UltraPure DNase/RNase-Free Distilled Water (Invitrogen, cat. no. 10977-023) UltraPure 10X TBE Buffer (Invitrogen, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 10,000x SYBR Safe DNA stain (Invitrogen, cat. no. ) Low DNA Mass Ladder (Invitrogen, cat. no. 10068-013) 1 kb Plus DNA Ladder (Invitrogen, cat. no. 10787-018) TrackIt™ Cyan/Orange Loading Buffer (Invitrogen, cat. no. 10482-028) Restriction enzymes: BsmBI ( Esp3I ) (Fermentas/ThermoScientific cat. no. ER0451) BsaI -HF (New England Biolabs, cat. no. R3535L) AfeI (New England Biolabs, cat. no. R0652S) Fermentas Tango Buffer and 10x NEBuffer 4 (included with enzymes) 100x Bovine Serum Albumin (New England Biolabs, included with Bsa I-HF) DL-Dithiothreitol (DTT) (Fermentas/ThermoScientific cat. no. R0862) T7 DNA ligase, 3,000 U/ul (Enzymatics, cat. no. L602L) CRITICAL Do not substitute the more commonly-used T4 ligase. .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354)

    Injection:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. .. Tryptic peptides were loaded onto a HPLC Chip (160 nl high-capacity sample enrichment column and 75 μ m × 150 mm SB-C18 separation column; Agilent Technologies, Santa Clara, CA, USA) and separated by flow rate at 300 nl per minute, with solvent A (0.2% (v/v) formic acid in water) and solvent B (100% acetonitrile) and the following gradients: at 0, 50, 54 and 56 min after injection with 3, 35, 80 and 100% solvent B, respectively.

    Recombinant:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Cleavage Assay:

    Article Title: Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
    Article Snippet: Paragraph title: Ub-AMC and ubiquitin cleavage assay ... Substrates and enzymes were incubated in Ub-AMC assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mg/ml ovalbumin [RPI Corp, 9006-59-1], 1 mM EDTA, 5 mM MgCl2 , 1 mM ATP [New England BioLabs, P0756L]).

    Multiplexing:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler. .. E. Adapter ligation: The mixture of 25 μL A-tailed DNA, 1 μL of 50 μmol/L multiplexing adapter, 3 μL of 10× T4 DNA ligase buffer (NEB, Cat. B0202), and 1 μL of 400 units/μL T4 DNA ligase (NEB, Cat. M0202L) was used in adapter ligation step followed by incubation at 16 °C overnight.

    Marker:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Magnetic Beads:

    Article Title: Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome
    Article Snippet: Reagents Molecular biology grade water (Corning, cat. no. 46–000-CM) Oligonucleotides and primers (IDT), see Table 25 mM MgCl2 (Life Technologies, cat. no. R0971) 10 mM Tris-EDTA (TE) buffer, pH 8.0 (Quality Biological, cat. no. 351–011-131) 1 M Tris-HCl buffer, pH 8.0 (Quality Biological, cat. no. 351–007-101) Ethanol, Absolute (200 Proof), Molecular Biology Grade (Fisher Scientific, cat. no. BP2818500) (CAUTION Ethanol is highly flammable) Ase I (NEB, cat. no. R0526S) BspH I (NEB, cat. no. R0517S) BstY I (NEB, cat. no. R0523S) Hind III (NEB, cat. no. R0104S) Nco I (NEB, cat. no. R0193S) Pst I (NEB, cat. no. R0140S) RNase cocktail enzyme mix (Life Technologies, cat. no. AM2286) T4 DNA ligase (NEB, cat. no. M0202S) Adenosine 5′-Triphosphate, ATP (NEB, cat. no. P0756S) TaKaRa Ex Taq DNA polymerase, Hot-Start (Clontech, cat. no. RR006A) QiaQuick PCR Purification Kit (Qiagen, cat. no. 28106) Zymoclean Gel DNA Recovery Kit (Zymo Research, cat. no D4002) Ultrapure Agarose (Life Technologies, cat. no. 16500–100) Gel Loading Dye, 6x (NEB, cat. no. B7022S) UltraPure Tris-Acetate-EDTA (TAE) buffer, 10x (Life Technologies, cat. no. 15558–026) Ethidium Bromide solution, 10 mg/mL (Bio-Rad, cat. no. 161–0433) (CAUTION Ethidium bromide is toxic and is a potential mutagen and carcinogen.) .. 2-log ladder (NEB, cat. no. N3200S) Qubit dsDNA HS assay kit (ThermoFisher Scientific, cat. no. Q32851) Agilent DNA 1000 kit (Agilent, cat. no. 5067–1504) Agencourt AMPure XP Magnetic Beads (Beckman Coulter, cat. no. A63882) KAPA HTP Library Preparation Kit for Illumina (KAPA Biosystems, cat. no. KK8234).

    Mutagenesis:

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: Phage display screening The ubiquitin variant library was constructed using oligonucleotide-directed mutagenesis ( ). .. The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs).

    Size-exclusion Chromatography:

    Article Title: Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes
    Article Snippet: A total reaction volume of 50 µL per sample was prepared on ice as follows: 16 mM HEPES-KOH, pH 7.6, 6.55 mg/mL creatine phosphate (10621722001; Roche), 0.1 mg/mL creatine kinase (10127566001; Roche), 0.8 mM ATP (P0756S; New England BioLabs), 0.1 mM spermidine, 0.1 mM complete amino acid mix (L4461; Promega), 135 mM potassium acetate (for purified mRNPs) or 50 mM potassium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), 1.5 mM magnesium acetate (for purified mRNP) or 2.5 mM magnesium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), and 20 µL of equilibrated (10 mg/mL) translation-competent cytoplasmic extract. .. A 6 µL aliquot was reserved in a prechilled microcentrifuge tube on ice as time point 0 and the remaining reaction directly transferred to a 37°C ThermoMixer (5382000015; Eppendorf) with ThermoTop lid (5308000003; Eppendorf) set to shake at 1500 rpm for 15 sec every 2 min. At indicated time points, 6 µL aliquots were taken and placed into prechilled microcentrifuge tubes on ice as conducted at time point 0.

    Electrophoretic Mobility Shift Assay:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. ..

    Purification:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Column purification of PCR product: All PCR products were purified using DNA Purification Kit (TIANGEN, Beijing, China, Cat. DP214-03). .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Article Title: Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes
    Article Snippet: .. A total reaction volume of 50 µL per sample was prepared on ice as follows: 16 mM HEPES-KOH, pH 7.6, 6.55 mg/mL creatine phosphate (10621722001; Roche), 0.1 mg/mL creatine kinase (10127566001; Roche), 0.8 mM ATP (P0756S; New England BioLabs), 0.1 mM spermidine, 0.1 mM complete amino acid mix (L4461; Promega), 135 mM potassium acetate (for purified mRNPs) or 50 mM potassium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), 1.5 mM magnesium acetate (for purified mRNP) or 2.5 mM magnesium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), and 20 µL of equilibrated (10 mg/mL) translation-competent cytoplasmic extract. .. Following the final wash in HLB150 + 0.1% NP-40, purified mRNPs were washed once in translation wash buffer (10 mM HEPES-KOH, pH 7.6, 135 mM potassium acetate, 1.5 mM magnesium acetate) then immediately resuspended in the 50 µL of prepared translation reaction.

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs). .. The double-stranded DNA was purified using a QIAquick DNA purification kit and transformed into Escherichia coli SS320 cells preinfected with M13KO7 helper phage.

    Article Title: Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome
    Article Snippet: .. Reagents Molecular biology grade water (Corning, cat. no. 46–000-CM) Oligonucleotides and primers (IDT), see Table 25 mM MgCl2 (Life Technologies, cat. no. R0971) 10 mM Tris-EDTA (TE) buffer, pH 8.0 (Quality Biological, cat. no. 351–011-131) 1 M Tris-HCl buffer, pH 8.0 (Quality Biological, cat. no. 351–007-101) Ethanol, Absolute (200 Proof), Molecular Biology Grade (Fisher Scientific, cat. no. BP2818500) (CAUTION Ethanol is highly flammable) Ase I (NEB, cat. no. R0526S) BspH I (NEB, cat. no. R0517S) BstY I (NEB, cat. no. R0523S) Hind III (NEB, cat. no. R0104S) Nco I (NEB, cat. no. R0193S) Pst I (NEB, cat. no. R0140S) RNase cocktail enzyme mix (Life Technologies, cat. no. AM2286) T4 DNA ligase (NEB, cat. no. M0202S) Adenosine 5′-Triphosphate, ATP (NEB, cat. no. P0756S) TaKaRa Ex Taq DNA polymerase, Hot-Start (Clontech, cat. no. RR006A) QiaQuick PCR Purification Kit (Qiagen, cat. no. 28106) Zymoclean Gel DNA Recovery Kit (Zymo Research, cat. no D4002) Ultrapure Agarose (Life Technologies, cat. no. 16500–100) Gel Loading Dye, 6x (NEB, cat. no. B7022S) UltraPure Tris-Acetate-EDTA (TAE) buffer, 10x (Life Technologies, cat. no. 15558–026) Ethidium Bromide solution, 10 mg/mL (Bio-Rad, cat. no. 161–0433) (CAUTION Ethidium bromide is toxic and is a potential mutagen and carcinogen.) .. 2-log ladder (NEB, cat. no. N3200S) Qubit dsDNA HS assay kit (ThermoFisher Scientific, cat. no. Q32851) Agilent DNA 1000 kit (Agilent, cat. no. 5067–1504) Agencourt AMPure XP Magnetic Beads (Beckman Coulter, cat. no. A63882) KAPA HTP Library Preparation Kit for Illumina (KAPA Biosystems, cat. no. KK8234).

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27106) QIAquick 96 PCR Purification (Qiagen, cat. no. 28181) UltraPure DNase/RNase-Free Distilled Water (Invitrogen, cat. no. 10977-023) UltraPure 10X TBE Buffer (Invitrogen, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 10,000x SYBR Safe DNA stain (Invitrogen, cat. no. ) Low DNA Mass Ladder (Invitrogen, cat. no. 10068-013) 1 kb Plus DNA Ladder (Invitrogen, cat. no. 10787-018) TrackIt™ Cyan/Orange Loading Buffer (Invitrogen, cat. no. 10482-028) Restriction enzymes: BsmBI ( Esp3I ) (Fermentas/ThermoScientific cat. no. ER0451) BsaI -HF (New England Biolabs, cat. no. R3535L) AfeI (New England Biolabs, cat. no. R0652S) Fermentas Tango Buffer and 10x NEBuffer 4 (included with enzymes) 100x Bovine Serum Albumin (New England Biolabs, included with Bsa I-HF) DL-Dithiothreitol (DTT) (Fermentas/ThermoScientific cat. no. R0862) T7 DNA ligase, 3,000 U/ul (Enzymatics, cat. no. L602L) CRITICAL Do not substitute the more commonly-used T4 ligase. .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354)

    Sequencing:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Paragraph title: Library preparation and sequencing for amplicons ... C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: Eppendorf tubes (1.5 ml) Pipette tips (10 μl, 100 μl, 1,000 μl) Deoxyguanosine triphosphate (dGTP) (New England Biolabs, catalog number: N0447S) 8-oxo-2′-deoxyguanosine-5′-Triphosphate (8-oxo-dGTP) (Jena Bioscience, catalog number: NU-1117L) DNA substrate: The DNA substrate includes a fluorescent tag at both the 5′- and 3′-ends of one of the gap-containing strand in the double-stranded DNA with a single nucleotide gap opposite template base Cytosine ( ) Note: The sequence information for the DNA substrate is presented in the Notes section of the protocol. .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes)

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs). .. The phosphorylated oligonucleotides were annealed to single-strand template DNA (20 μg) containing the wild-type ubiquitin sequence fused with the M13 major coat protein P3 [described by McLaughlin and Sidhu ( )] by incubating at 90°C for 3 min, at 50°C for 3 min, and at 20°C for 5 min. Complementary DNA primed by the phospho-oligonucleotides was synthesized and ligated by the addition of 10 μl of 10 mM ATP, 10 μl of a 10 mM deoxynucleotide triphosphate mixture, 15 μl of 100 mM DTT, 30 Weiss units of T4 DNA ligase, and 30 U of T7 DNA at 20°C overnight.

    Ub-AMC Assay:

    Article Title: Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
    Article Snippet: .. Substrates and enzymes were incubated in Ub-AMC assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mg/ml ovalbumin [RPI Corp, 9006-59-1], 1 mM EDTA, 5 mM MgCl2 , 1 mM ATP [New England BioLabs, P0756L]). .. After adding of Ub-AMC, the reaction was initiated, and an Envision plate reader (PerkinElmer, MA, USA) was used to measure mixture at Ex345/Em445.

    Polyacrylamide Gel Electrophoresis:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Staining:

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27106) QIAquick 96 PCR Purification (Qiagen, cat. no. 28181) UltraPure DNase/RNase-Free Distilled Water (Invitrogen, cat. no. 10977-023) UltraPure 10X TBE Buffer (Invitrogen, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 10,000x SYBR Safe DNA stain (Invitrogen, cat. no. ) Low DNA Mass Ladder (Invitrogen, cat. no. 10068-013) 1 kb Plus DNA Ladder (Invitrogen, cat. no. 10787-018) TrackIt™ Cyan/Orange Loading Buffer (Invitrogen, cat. no. 10482-028) Restriction enzymes: BsmBI ( Esp3I ) (Fermentas/ThermoScientific cat. no. ER0451) BsaI -HF (New England Biolabs, cat. no. R3535L) AfeI (New England Biolabs, cat. no. R0652S) Fermentas Tango Buffer and 10x NEBuffer 4 (included with enzymes) 100x Bovine Serum Albumin (New England Biolabs, included with Bsa I-HF) DL-Dithiothreitol (DTT) (Fermentas/ThermoScientific cat. no. R0862) T7 DNA ligase, 3,000 U/ul (Enzymatics, cat. no. L602L) CRITICAL Do not substitute the more commonly-used T4 ligase. .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354)

    Concentration Assay:

    Article Title: IDH3α regulates one-carbon metabolism in glioblastoma
    Article Snippet: These primers were then added at 0.5 μM concentration to 100 ng of IDH3α vector DNA along with 200 μM of deoxynucleotide triphosphate, 1.25 U of PrimeSTAR HS (catalog no. R010A, Takara), and PrimeSTAR 5× Buffer to obtain a 1× final concentration. .. The product band was excised, extracted, and treated with 10 U of T4 PNK (catalog no. M0201, New England Biolabs) in 1× of T4 PNK buffer (catalog no. B0201, New England Biolabs), 1 mM ATP (catalog no. P0756, New England Biolabs), and nuclease-free water (total volume, 50 μl).

    Chromatin Immunoprecipitation:

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. .. Tryptic peptides were loaded onto a HPLC Chip (160 nl high-capacity sample enrichment column and 75 μ m × 150 mm SB-C18 separation column; Agilent Technologies, Santa Clara, CA, USA) and separated by flow rate at 300 nl per minute, with solvent A (0.2% (v/v) formic acid in water) and solvent B (100% acetonitrile) and the following gradients: at 0, 50, 54 and 56 min after injection with 3, 35, 80 and 100% solvent B, respectively.

    SDS Page:

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. In-gel digestion and mass spectrometry analysis The kinase assay as indicated above was repeated, replacing the radiolabelled γ32 P-ATP with unlabeled ATP (NEB, P0756S), and the band shift was observed once again in a Coomassie blue-stained gel when GST-Fra-2 was incubated with ERK 2; however, an 8% SDS-PAGE was run to achieve greater resolution of the bands. ..

    Plasmid Preparation:

    Article Title: IDH3α regulates one-carbon metabolism in glioblastoma
    Article Snippet: These primers were then added at 0.5 μM concentration to 100 ng of IDH3α vector DNA along with 200 μM of deoxynucleotide triphosphate, 1.25 U of PrimeSTAR HS (catalog no. R010A, Takara), and PrimeSTAR 5× Buffer to obtain a 1× final concentration. .. The product band was excised, extracted, and treated with 10 U of T4 PNK (catalog no. M0201, New England Biolabs) in 1× of T4 PNK buffer (catalog no. B0201, New England Biolabs), 1 mM ATP (catalog no. P0756, New England Biolabs), and nuclease-free water (total volume, 50 μl).

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354) .. HEK293FT cells (Invitrogen, cat. no. R700-07) Dulbecco’s Minimum Eagle Medium (DMEM) (1X), high glucose (Invitrogen, cat. no. 10313-039) Dulbecco’s Phosphate Buffered Saline (DPBS) (1X) (Invitrogen, cat. no. 14190-250) Fetal bovine serum, qualified and heat inactivated (Invitrogen, cat. no. 10438-034) Opti-MEM® I reduced-serum medium (Invitrogen, cat. no. 11058-021) GlutaMAX™-I (100X) (Invitrogen, cat. no. 35050079) Penicillin-streptomycin (100X) (Invitrogen, cat. no. 15140-163) Trypsin, 0.05% (1X) with EDTA•4Na (Invitrogen, cat. no. 25300-062) Lipofectamine 2000 ™ transfection reagent (Invitrogen, cat. no. 11668027) QuickExtract ™ DNA extraction solution (Epicentre, cat. no. QE09050) Herculase II Fusion polymerase (Agilent Technologies, cat. no. 600679) CRITICAL Since Surveyor assay is sensitive to single-base mismatches, it is important to use only a high-fidelity polymerase.

    Multiplex Assay:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Library preparation and sequencing for amplicons The detailed protocol of target deep DNA sequencing was as follows: A. Multiplex PCR amplification: The 128 amplicons were amplified by multiplex PCR on Veriti 96-well Thermal Cycler (Applied Biosystems, Waltham, MA, USA), which was performed using 30 ng genomic DNA, 15 μL Primer mix/pool (2 pools in total), 10 μL Q5 reaction buffer (NEB, Ipswich, MA, USA, Cat. B9027S), 10 μL Q5 high GC enhancer (NEB, Cat. B9028A), 1.5 μL dNTPs mix (NEB, Cat. N0447S), 0.5 μL Q5 high-fidelity DNA polymerase (NEB, Cat. M0491L), and ddH2 O to make the final reaction volume to 50 μL. .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    In Vitro:

    Article Title: Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes
    Article Snippet: Paragraph title: In vitro translation ... A total reaction volume of 50 µL per sample was prepared on ice as follows: 16 mM HEPES-KOH, pH 7.6, 6.55 mg/mL creatine phosphate (10621722001; Roche), 0.1 mg/mL creatine kinase (10127566001; Roche), 0.8 mM ATP (P0756S; New England BioLabs), 0.1 mM spermidine, 0.1 mM complete amino acid mix (L4461; Promega), 135 mM potassium acetate (for purified mRNPs) or 50 mM potassium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), 1.5 mM magnesium acetate (for purified mRNP) or 2.5 mM magnesium acetate (for IVT mRNA and protein-stripped PP7-purified mRNA), and 20 µL of equilibrated (10 mg/mL) translation-competent cytoplasmic extract.

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: Paragraph title: In vitro kinase reactions ... Immunoprecipitation used either protein G–Sepharose or GSH beads, and these were then washed three times with kinase wash buffer (25 m m Tris-HCl, pH 7.5, 70 m m NaCl, 10 m m MgCl2 , 1 m m DTT) and resuspended in kinase reaction buffer (kinase wash buffer, 10 μ m ATP (New England Biolabs, P0756), [γ-32 P]ATP for phosphorylation of full-length SF3B1 only (5 μCi/reaction; 1 Ci = 37 GBq), 0.5 μg of active cyclin E-CDK2 (Millipore, 14-475) followed by incubation at 37 °C.

    Immunoprecipitation:

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Immunoprecipitation used either protein G–Sepharose or GSH beads, and these were then washed three times with kinase wash buffer (25 m m Tris-HCl, pH 7.5, 70 m m NaCl, 10 m m MgCl2 , 1 m m DTT) and resuspended in kinase reaction buffer (kinase wash buffer, 10 μ m ATP (New England Biolabs, P0756), [γ-32 P]ATP for phosphorylation of full-length SF3B1 only (5 μCi/reaction; 1 Ci = 37 GBq), 0.5 μg of active cyclin E-CDK2 (Millipore, 14-475) followed by incubation at 37 °C. .. Synchronized HeLa cells were resuspended in TRIzol (Thermo Fisher Scientific, 15596026).

    DNA Purification:

    Article Title: Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells
    Article Snippet: Column purification of PCR product: All PCR products were purified using DNA Purification Kit (TIANGEN, Beijing, China, Cat. DP214-03). .. C. End repair and A-tailing of DNA fragments: The mixture of 37.5 μL DNA, 5 μL Cut Smart (NEB, Cat. B7204S), 5 μL Adenosine 5′-Triphosphate (NEB, Cat. P0756L), 0.5 μL of 100 mmol/L dATP solution (NEB, Cat. N0440S), 1 μL T4 Polynucleotide Kinase (NEB, Cat. M0201L) and 1 μL 5 units Klenow exo-DNA polymerase (NEB, Cat. M0212L) was incubated at 37 °C for 1 h on Veriti 96-well Thermal Cycler.

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs). .. The double-stranded DNA was purified using a QIAquick DNA purification kit and transformed into Escherichia coli SS320 cells preinfected with M13KO7 helper phage.

    Lysis:

    Article Title: Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia
    Article Snippet: .. Rat hippocampal tissues were homogenized in lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP40, 1 mM DTT, 2 mM ATP [New England Biolabs, P0756S]). .. Lysate (250 µl) containing 3 μg of protein were incubated with 40 nM aminomethylcoumarin (AMC)-linked synthetic peptide substrates for 30 min at 37°C.

    High Throughput Screening Assay:

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples
    Article Snippet: Paragraph title: High-throughput CUTseq ... After incubating the plate at 37 °C for 30 min, we used I-DOT again to dispense 300 nl per well of CUTseq adapter at 33 nM, using a differently barcoded adapter for each well, followed by 200 nl of T4 rapid DNA ligase (Thermo Fisher Scientific, catalog number K1423), 300 nl of T4 ligase buffer 5× (Thermo Fisher Scientific, catalog number K1423), 120 nl of ATP at 10 μΜ (NEB, catalog number P0756L), 30 nl of 50 mg/ml bovine serum albumin (Thermo Fisher Scientific, catalog number AM2616), and 50 nl of nuclease-free water (Thermo Fisher Scientific, catalog number 4387936).

    Gel Extraction:

    Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering
    Article Snippet: 5x Herculase II reaction buffer (Agilent Technologies, included with polymerase) Taq-B polymerase (Enzymatics, cat. no. P725L) 10x Taq-B buffer (Enzymatics, included with polymerase) 25 mM (each) dNTP solution mix (Enzymatics, cat. no. N205L) MinElute Gel Extraction Kit (Qiagen, cat. no. 28606) CRITICAL MinElute columns should be stored at 4°C until use. .. 10 mM Adenosine 5′-Triphosphate (New England Biolabs, cat. no. P0756S) Plasmid-Safe™ ATP-Dependent DNase (Epicentre, cat. no. E3101K) One Shot® Stbl3™ Chemically Competent E. coli (Invitrogen, cat. no. C7373-03) SOC medium (New England Biolabs, cat. no. B9020S) LB medium (Sigma, cat. no. L3022) LB agar medium (Sigma, cat. no. L2897) 100 mg/ml ampicillin, sterile-filtered (Sigma, cat. no. A5354)

    Variant Assay:

    Article Title: Protein engineering by highly parallel screening of computationally designed variants
    Article Snippet: Phage display screening The ubiquitin variant library was constructed using oligonucleotide-directed mutagenesis ( ). .. The oligonucleotide library (0.6 μg) was 5′-phosphorylated for 1 hour at 37°C in TM buffer [10 mM MgCl2 , 50 mM tris-HCl (pH 7.5)] with 1 mM adenosine 5′-triphosphate (ATP), 5 mM dithiothreitol (DTT), using a T4 polynucleotide kinase (1 U/μl; New England Biolabs).

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  • 79
    New England Biolabs α β methylene atp
    The ratios of phosphorylated to total MAPK 42/44 of three individual preparations demonstrate concentration-dependent activation of MAPK 42/44 after 10 min <t>ATP</t> or UTP. α - β -Methylene-ATP and UTP failed to stimulate MAPK 42/44 . The ratio after 10 min without agonists was used as a control and set to 100%.
    α β Methylene Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs γ 32 p atp
    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 <t>P]ATP,</t> followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.
    γ 32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The ratios of phosphorylated to total MAPK 42/44 of three individual preparations demonstrate concentration-dependent activation of MAPK 42/44 after 10 min ATP or UTP. α - β -Methylene-ATP and UTP failed to stimulate MAPK 42/44 . The ratio after 10 min without agonists was used as a control and set to 100%.

    Journal: British Journal of Pharmacology

    Article Title: P2Y-Receptors stimulating the proliferation of human mesangial cells through the MAPK42/44 pathway

    doi: 10.1038/sj.bjp.0705358

    Figure Lengend Snippet: The ratios of phosphorylated to total MAPK 42/44 of three individual preparations demonstrate concentration-dependent activation of MAPK 42/44 after 10 min ATP or UTP. α - β -Methylene-ATP and UTP failed to stimulate MAPK 42/44 . The ratio after 10 min without agonists was used as a control and set to 100%.

    Article Snippet: Kinetics of MAPK42/44 phosphorylation were done by stimulation of resting mesangial cells plated on six-well plates with ATP (100 μ M ) for 0, 2, 5, 10, 15, 20 and 60 min. To determinate concentration dependency, various concentrations of ATP, α - β -methylene-ATP and UTP were applied on the cells for 10 min. For experiments using the mitogen-activated protein (MAP) kinase (MEK) inhibitor PD-98059 (New England BioLabs, Beverly, U.S.A.), cells were incubated for 30 min at 37°C in growth-arresting medium that contained the inhibitor before the addition of agonists.

    Techniques: Concentration Assay, Activation Assay

    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Journal: Cancer research

    Article Title: Plk1 Phosphorylation of Mre11 Antagonizes the DNA Damage Response

    doi: 10.1158/0008-5472.CAN-16-2787

    Figure Lengend Snippet: Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Article Snippet: After the 5′ end of TP423 was labelled with [γ-32 P]ATP and polynucleotide kinase (New England Biolabs), TP423 and TP424 oligonucleotides were annealed to generate the 3′ overhanging DNA duplexes.

    Techniques: Incubation, Immunoprecipitation, Avidin-Biotin Assay, Lysis, Purification, Autoradiography, In Vitro, SDS Page, Staining, In Vivo, Transfection, Construct

    FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: In Vivo, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Immunoprecipitation, Variant Assay, Labeling, Polymerase Chain Reaction

    FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Transfection, Western Blot, RNA Binding Assay, Expressing

    In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: In Vitro, Mutagenesis, Construct, Reverse Transcription Polymerase Chain Reaction, Marker, Labeling, Agarose Gel Electrophoresis

    RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Labeling, Marker