Structured Review

Thermo Fisher fastap
Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM <t>MDCC-PBP,</t> 0.0004u/μl <t>FastAP</t> and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
Fastap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 103 article reviews
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Images

1) Product Images from "Quantitative Microplate Assay for Real-Time Nuclease Kinetics"

Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0154099

Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
Figure Legend Snippet: Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

Techniques Used: Concentration Assay, Phosphate Release Assay, Incubation, Fluorescence, De-Phosphorylation Assay, Binding Assay, Derivative Assay

2) Product Images from "Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7"

Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky644

Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.
Figure Legend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

Techniques Used: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation

3) Product Images from "Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7"

Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky644

Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.
Figure Legend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

Techniques Used: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation

Related Articles

DNA Extraction:

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

Nucleic Acid Electrophoresis:

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

Mutagenesis:

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

Purification:

Article Title: The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis
Article Snippet: .. SBE products were purified using FastApp Thermosensitive Alkaline Phosphatase (Fermentas) according to the manufacturer's protocol. .. Detection of SNP products was done on an ABI PRISM™ 3130 Genetic Analyzer (Applied Biosystems) using 2 μl purified SNaPshot product in 11.7 μl HD formamide and 0.3 μl LIZ120 standard (all Applied Biosystems).

Article Title: Response to IL‐17A inhibitors secukinumab and ixekizumab cannot be explained by genetic variation in the protein‐coding and untranslated regions of the IL‐17A gene: results from a multicentre study of four European psoriasis cohorts
Article Snippet: .. PCR products were purified with FastAP Thermosensitive Alkaline Phosphatase and Exonuclease I (Thermo Fisher Scientific, Waltham, MA, USA) and the IL‐17A gene was Sanger sequenced, using the PCR primers described previously. .. Sequence results were analysed using Vector NTI software from Thermo Fisher Scientific.

Polymerase Chain Reaction:

Article Title: Spotted fever Rickettsia species in Hyalomma and Ixodes ticks infesting migratory birds in the European Mediterranean area
Article Snippet: .. All PCR products considered for sequencing were cleaned using Exonuclease I and FastAPTM Thermosensitive Alkaline Phosphatase (Fermentas GmbH). .. Direct cycle sequencing of PCR products from 2009 year’s samples was performed using BigDye® Terminator v 3.1 Cycle Sequencing Kit in an ABI 3130 instrument (Applied Biosystems).

Article Title: Positive Selection and Functional Divergence at Meiosis Genes That Mediate Crossing Over Across the Drosophila Phylogeny
Article Snippet: .. The 5′ end of the resulting PCR product was digested with SphI (New England Biolabs, Ipswich, MA) while the 3′ end was made blunt using FastAp Thermosenstive Alkaline Phosphatase (Thermo Fisher, Waltham, MA). .. This fragment was cloned into the SphI /SpeI sites of KS+, generating intermediate plasmid KS[virfrag1 ].

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

Article Title: Response to IL‐17A inhibitors secukinumab and ixekizumab cannot be explained by genetic variation in the protein‐coding and untranslated regions of the IL‐17A gene: results from a multicentre study of four European psoriasis cohorts
Article Snippet: .. PCR products were purified with FastAP Thermosensitive Alkaline Phosphatase and Exonuclease I (Thermo Fisher Scientific, Waltham, MA, USA) and the IL‐17A gene was Sanger sequenced, using the PCR primers described previously. .. Sequence results were analysed using Vector NTI software from Thermo Fisher Scientific.

Article Title: Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes
Article Snippet: .. Following treatment with alkaline phosphatase (FastAP; EF0651; Fermentas) and exonuclease I (EN0581; Fermentas), the PCR product was sequenced directly with KanB via standard dideoxy methods to verify the gene-specific barcode within this region ( ). ..

Sequencing:

Article Title: Spotted fever Rickettsia species in Hyalomma and Ixodes ticks infesting migratory birds in the European Mediterranean area
Article Snippet: .. All PCR products considered for sequencing were cleaned using Exonuclease I and FastAPTM Thermosensitive Alkaline Phosphatase (Fermentas GmbH). .. Direct cycle sequencing of PCR products from 2009 year’s samples was performed using BigDye® Terminator v 3.1 Cycle Sequencing Kit in an ABI 3130 instrument (Applied Biosystems).

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

Staining:

Article Title: Genome engineering using the CRISPR-Cas9 system
Article Snippet: .. PCR primers for SURVEYOR, RFLP analysis or sequencing; see , (alternatively, they can be custom made) QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) TBE Gels, 4–20%, 1.0 mm, 15 well (Life Technologies, cat. no. C62255BOX) Novex Hi-Density TBE sample buffer, 5× (Life Technologies, cat. no. LC6678) SYBR Gold nucleic acid gel stain, 10,000× (Life Technologies, cat. no. S-11494) FastDigest HindIII (Fermentas/Thermo Scientific, cat. no. FD0504) FastDigest buffer, 10× (Fermentas/Thermo Scientific, supplied with FastDigest HindIII) FastAP Antarctic phosphatase (Fermentas/Thermo Scientific, cat. no. EF0654) Nextera XT index kit (Illumina, cat. no. FC-131-1001) .. Filtered sterile pipette tips (Corning) Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. no. 0030 125.150) Axygen PCR plates, 96 well (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070) Round-bottom tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes, 60 mm × 15 mm (BD Biosciences, cat. no. 351007) Tissue culture plate, 24 wells (BD Falcon, cat. no. 353047) Tissue culture plate, 96 wells flat bottom (BD Falcon, cat. no. 353075) Tissue culture dish, 100 mm (BD Falcon, cat. no. 353003) Nunc EasYFlask 225 cm2 (T225 flask), filter cap, 70-ml working volume (Thermo Scientific, cat. no. 159934) Nunc EasYFlask 75 cm2 (T75 flask), filter cap, 25-ml working volume (Thermo Scientific, cat. no. 156499) INCYTO C-Chip disposable hemocytometer (VWR, cat. no. 82030-468) Steriflip-GP Filter Unit, 0.22 μM (Millipore, cat. no. SCGP00525) Thermocycler with programmable temperature stepping functionality, 96 well (Applied Biosystems Veriti, cat. no. 4375786) Desktop microcentrifuges (e.g., Eppendorf, cat. nos.

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    Thermo Fisher dbsnp sap refseq crap fasta files
    Comparison of average XCorr scores for peptides matching the <t>RefSeq</t> protein, <t>dbSNP-SAP,</t> or custom (RNA-Seq) SAP database. SAP peptides identified from the custom database tended to have higher XCorr scores than those identified from the dbSNP database.
    Dbsnp Sap Refseq Crap Fasta Files, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dbsnp sap refseq crap fasta files/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dbsnp sap refseq crap fasta files - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    92
    Thermo Fisher fastap
    Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM <t>MDCC-PBP,</t> 0.0004u/μl <t>FastAP</t> and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
    Fastap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fastap/product/Thermo Fisher
    Average 92 stars, based on 103 article reviews
    Price from $9.99 to $1999.99
    fastap - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Comparison of average XCorr scores for peptides matching the RefSeq protein, dbSNP-SAP, or custom (RNA-Seq) SAP database. SAP peptides identified from the custom database tended to have higher XCorr scores than those identified from the dbSNP database.

    Journal: Journal of proteome research

    Article Title: Large-scale mass spectrometric detection of variant peptides resulting from non-synonymous nucleotide differences

    doi: 10.1021/pr4009207

    Figure Lengend Snippet: Comparison of average XCorr scores for peptides matching the RefSeq protein, dbSNP-SAP, or custom (RNA-Seq) SAP database. SAP peptides identified from the custom database tended to have higher XCorr scores than those identified from the dbSNP database.

    Article Snippet: Raw mass spectrometry files were searched against the customized SAP+RefSeq+cRAP and the dbSNP-SAP+RefSeq+cRAP FASTA files using the SEQUEST/Percolator search algorithm within ProteomeDiscoverer (v1.3.0.339, Thermo Fisher Scientific, San Jose, CA).

    Techniques: RNA Sequencing Assay

    Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Journal: PLoS ONE

    Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    doi: 10.1371/journal.pone.0154099

    Figure Lengend Snippet: Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Article Snippet: MDCC-PBP (commercial name Phosphate Sensor), T4 polynucleotide kinase, FastAP and Exonuclease III (ThermoFisher).

    Techniques: Concentration Assay, Phosphate Release Assay, Incubation, Fluorescence, De-Phosphorylation Assay, Binding Assay, Derivative Assay

    Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Article Snippet: DNase treatment and purification of RNA fragments To obtain the purified, dephosphorylated native fragment, the DNA/RNA hybrid solution was incubated with 0.1 U/μl FastAP (Thermo Scientific) and 1.25 U/μl DNaseI (Thermo Scientific) for 1 h at 37°C.

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation

    Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Article Snippet: To obtain the purified, dephosphorylated native fragment, the DNA/RNA hybrid solution was incubated with 0.1 U/μl FastAP (Thermo Scientific) and 1.25 U/μl DNaseI (Thermo Scientific) for 1 h at 37°C.

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation