fastap thermosensitive alkaline phosphatase  (Thermo Fisher)


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    Name:
    FastAP Thermosensitive Alkaline Phosphatase
    Description:
    Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA, and nucleotides. This enzyme also removes phosphate groups from proteins.FastAP is a novel alkaline phosphatase, which is active in all Thermo Scientific restriction enzyme buffers as well as in PCR buffers. It dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs) in 10 minutes at 37°C. The enzyme is inactivated in 5 minutes at 75°C (see Figure 1 in Supporting Data). Therefore, removal of alkaline phosphatase is not required prior to ligation.Highlights• Recombinant enzyme• Fast dephosphorylation—10 minutes at 37°C• Fast and complete inactivation—5 minutes at 75°C• Simultaneous digestion and dephosphorylation of vector DNA• 100% active in restriction enzyme and PCR buffers• PCR clean-up in conjunction with Exo I• Protein dephosphorylationOne protocol for all types of DNA ends:• 5'-overhangs• 3'-overhangs• blunt-ends• single nucleotidesApplications• Dephosphorylation of cloning vector DNA to prevent recircularization during ligation• Simultaneous digestion and dephosphorylation of vector DNA• PCR product clean-up: nucleotide degradation prior to sequencing of PCR product• Dephosphorylation of nucleic acid 5'-termini prior to labeling with T4 Polynucleotide Kinase• Other applications where dephosphorylation of DNA and RNA substrates is necessary• Protein dephosphorylationNote• Binding of FastAP Thermosensitive Alkaline Phosphatase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 65°C for 10 minutes, and chill on ice prior to electrophoresis.• FastAP Thermosensitive Alkaline Phosphatase is active in all restriction enzyme buffers and may be added directly to digested DNA. Heat inactivation of the restriction enzyme before dephosphorylation reaction is not necessary.
    Catalog Number:
    EF0651
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Size:
    1 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher fastap thermosensitive alkaline phosphatase
    Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA, and nucleotides. This enzyme also removes phosphate groups from proteins.FastAP is a novel alkaline phosphatase, which is active in all Thermo Scientific restriction enzyme buffers as well as in PCR buffers. It dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs) in 10 minutes at 37°C. The enzyme is inactivated in 5 minutes at 75°C (see Figure 1 in Supporting Data). Therefore, removal of alkaline phosphatase is not required prior to ligation.Highlights• Recombinant enzyme• Fast dephosphorylation—10 minutes at 37°C• Fast and complete inactivation—5 minutes at 75°C• Simultaneous digestion and dephosphorylation of vector DNA• 100% active in restriction enzyme and PCR buffers• PCR clean-up in conjunction with Exo I• Protein dephosphorylationOne protocol for all types of DNA ends:• 5'-overhangs• 3'-overhangs• blunt-ends• single nucleotidesApplications• Dephosphorylation of cloning vector DNA to prevent recircularization during ligation• Simultaneous digestion and dephosphorylation of vector DNA• PCR product clean-up: nucleotide degradation prior to sequencing of PCR product• Dephosphorylation of nucleic acid 5'-termini prior to labeling with T4 Polynucleotide Kinase• Other applications where dephosphorylation of DNA and RNA substrates is necessary• Protein dephosphorylationNote• Binding of FastAP Thermosensitive Alkaline Phosphatase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 65°C for 10 minutes, and chill on ice prior to electrophoresis.• FastAP Thermosensitive Alkaline Phosphatase is active in all restriction enzyme buffers and may be added directly to digested DNA. Heat inactivation of the restriction enzyme before dephosphorylation reaction is not necessary.
    https://www.bioz.com/result/fastap thermosensitive alkaline phosphatase/product/Thermo Fisher
    Average 99 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    fastap thermosensitive alkaline phosphatase - by Bioz Stars, 2019-12
    99/100 stars

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    Related Articles

    In Vitro:

    Article Title: Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains
    Article Snippet: RNAs for electrophoretic mobility shift assays (EMSA) were labeled radioactively for sensitive detection of protein–RNA interactions. .. In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA, 1x Tango buffer with BSA (Thermo Fisher), 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher). .. After incubation at 37 °C for 15 min, the dephosphorylated RNA was phenol/chloroform extracted and precipitated with 0.1 V 3 M NaOAc, 3 V absolute ethanol and subsequent chilling at −20 °C for ≥15 min. For radioactive labeling, 10 pmol dephosphorylated RNA or chemically synthesized RNA were 5′-phosphorylated with 32 P from γ-32 P ATP (Hartmann Analytic) in a 20 µL reaction with T4 polynucleotide kinase (New England Biolabs) in 1× buffer A.

    Labeling:

    Article Title: Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains
    Article Snippet: Paragraph title: Radioactive labeling of RNA ... In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA, 1x Tango buffer with BSA (Thermo Fisher), 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher).

    Purification:

    Article Title: Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains
    Article Snippet: In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA, 1x Tango buffer with BSA (Thermo Fisher), 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher). .. After incubation at 37 °C for 15 min, the dephosphorylated RNA was phenol/chloroform extracted and precipitated with 0.1 V 3 M NaOAc, 3 V absolute ethanol and subsequent chilling at −20 °C for ≥15 min. For radioactive labeling, 10 pmol dephosphorylated RNA or chemically synthesized RNA were 5′-phosphorylated with 32 P from γ-32 P ATP (Hartmann Analytic) in a 20 µL reaction with T4 polynucleotide kinase (New England Biolabs) in 1× buffer A.

    Article Title: The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis
    Article Snippet: RNA pull-down and input samples were incubated for 1 hr at 65°C in NLS digestion buffer (20 mM Tris-Cl pH 7.5, 10 mM EDTA, 2% N-lauroylsarcosine, 2.5 mM TCEP, 500 mM NaCl, and 250 μg proteinase K) to remove proteins and reverse the formaldehyde cross-links, followed by nucleic acid purification using Dynabeads MyOne Silane beads (Thermo Fisher Scientific). .. DNA was digested at 37°C for 30 min in a buffer containing 10 mM Tris-Cl pH 7.5, 1 mM MgCl2 , 120 μM CaCl2 , 10 mM KCl, 1 mM DTT, 0.002% Triton X-100, 40U murine RNase inhibitor (New England Biolabs), 3U FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific), 30U T4 polynucleotide kinase (New England Biolabs), 2U Turbo DNase (Thermo Fisher Scientific), and 20U Exonuclease I (New England Biolabs), followed by RNA purification using Dynabeads MyOne Silane beads. .. 20 pmol RiL-19 RNA adapter ( 5’-[phosph]rArGrArUrCrGrGrArArGrArGrCrGrUrCrGrUrG[3ddC] ) ( ) was ligated to the purified RNAs by incubation in ligation mix [1x T4 RNA ligase reaction 1 buffer, 9% DMSO, 1 mM ATP, 20% PEG8000 (New England Biolabs), 12U murine RNase inhibitor, and 40U T4 RNA ligase 1 (New England Biolabs)] for 1.5 hr at room temperature, followed by RNA purification using Dynabeads MyOne Silane beads.

    Concentration Assay:

    Article Title: Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains
    Article Snippet: In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA, 1x Tango buffer with BSA (Thermo Fisher), 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher). .. Remaining free nucleotides were removed by purification on a NucAway™ Spin column (Ambion) according to the manufacturer’s instructions.

    Incubation:

    Article Title: The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis
    Article Snippet: RNA pull-down and input samples were incubated for 1 hr at 65°C in NLS digestion buffer (20 mM Tris-Cl pH 7.5, 10 mM EDTA, 2% N-lauroylsarcosine, 2.5 mM TCEP, 500 mM NaCl, and 250 μg proteinase K) to remove proteins and reverse the formaldehyde cross-links, followed by nucleic acid purification using Dynabeads MyOne Silane beads (Thermo Fisher Scientific). .. DNA was digested at 37°C for 30 min in a buffer containing 10 mM Tris-Cl pH 7.5, 1 mM MgCl2 , 120 μM CaCl2 , 10 mM KCl, 1 mM DTT, 0.002% Triton X-100, 40U murine RNase inhibitor (New England Biolabs), 3U FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific), 30U T4 polynucleotide kinase (New England Biolabs), 2U Turbo DNase (Thermo Fisher Scientific), and 20U Exonuclease I (New England Biolabs), followed by RNA purification using Dynabeads MyOne Silane beads.

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: The bands corresponding to 40-100 nt were cut out and recovered using a ZR small-RNA PAGE recovery kit (Zymo research). .. The recovered RNAs were incubated in a dephosphorylation mixture containing 8 U FastAP thermosensitive alkaline phosphatase (Thermo Scientific, EF0651) and 40 U RNase inhibitors in polynucleotide kinase (PNK) buffer for 45 min at 20 °C. .. RNAs were subsequently phosphorylated with 10 U of T4 polynucleotide kinase in PNK buffer (TAKARA) for 30 min at 37 °C.

    Electrophoretic Mobility Shift Assay:

    Article Title: Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains
    Article Snippet: RNAs for electrophoretic mobility shift assays (EMSA) were labeled radioactively for sensitive detection of protein–RNA interactions. .. In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA, 1x Tango buffer with BSA (Thermo Fisher), 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher).

    De-Phosphorylation Assay:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: The bands corresponding to 40-100 nt were cut out and recovered using a ZR small-RNA PAGE recovery kit (Zymo research). .. The recovered RNAs were incubated in a dephosphorylation mixture containing 8 U FastAP thermosensitive alkaline phosphatase (Thermo Scientific, EF0651) and 40 U RNase inhibitors in polynucleotide kinase (PNK) buffer for 45 min at 20 °C. .. RNAs were subsequently phosphorylated with 10 U of T4 polynucleotide kinase in PNK buffer (TAKARA) for 30 min at 37 °C.

    Nucleic Acid Purification:

    Article Title: The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis
    Article Snippet: RNA pull-down and input samples were incubated for 1 hr at 65°C in NLS digestion buffer (20 mM Tris-Cl pH 7.5, 10 mM EDTA, 2% N-lauroylsarcosine, 2.5 mM TCEP, 500 mM NaCl, and 250 μg proteinase K) to remove proteins and reverse the formaldehyde cross-links, followed by nucleic acid purification using Dynabeads MyOne Silane beads (Thermo Fisher Scientific). .. DNA was digested at 37°C for 30 min in a buffer containing 10 mM Tris-Cl pH 7.5, 1 mM MgCl2 , 120 μM CaCl2 , 10 mM KCl, 1 mM DTT, 0.002% Triton X-100, 40U murine RNase inhibitor (New England Biolabs), 3U FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific), 30U T4 polynucleotide kinase (New England Biolabs), 2U Turbo DNase (Thermo Fisher Scientific), and 20U Exonuclease I (New England Biolabs), followed by RNA purification using Dynabeads MyOne Silane beads.

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  • 99
    Thermo Fisher fastap thermosensitive alkaline phosphatase 1 u µl
    Fastap Thermosensitive Alkaline Phosphatase 1 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fastap thermosensitive alkaline phosphatase 1 u µl/product/Thermo Fisher
    Average 99 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    fastap thermosensitive alkaline phosphatase 1 u µl - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

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