length fap265 gene  (New England Biolabs)


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    Name:
    M MuLV Reverse Transcriptase
    Description:
    M MuLV Reverse Transcriptase 50 000 units
    Catalog Number:
    m0253l
    Price:
    288
    Size:
    50 000 units
    Category:
    Reverse Transcriptases
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    New England Biolabs length fap265 gene
    M MuLV Reverse Transcriptase
    M MuLV Reverse Transcriptase 50 000 units
    https://www.bioz.com/result/length fap265 gene/product/New England Biolabs
    Average 93 stars, based on 465 article reviews
    Price from $9.99 to $1999.99
    length fap265 gene - by Bioz Stars, 2020-04
    93/100 stars

    Images

    1) Product Images from "Chlamydomonas FAP265 is a tubulin polymerization promoting protein, essential for flagellar reassembly and hatching of daughter cells from the sporangium"

    Article Title: Chlamydomonas FAP265 is a tubulin polymerization promoting protein, essential for flagellar reassembly and hatching of daughter cells from the sporangium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0185108

    Validation of fap265 mutant strain. (A) PCR amplification using FAP265 gene specific primers showed a specific band at ~2.0 kb with CC-4533 but not with fap265 mutant genomic DNA. (B) Equal amount of whole cell lysates from CC-4533 and fap265 mutant cells were separated on 12% SDS-PAGE; western blotting using antibodies against recombinant FAP265 detects a specific band at ~16.2 kDa in CC-4533 but not in fap265 lysates. Western blotting using antibodies against α-tubulin was used as loading control. (C) CC-4533 and fap265 cells were stained with antibodies against FAP265 (Red) and Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: Validation of fap265 mutant strain. (A) PCR amplification using FAP265 gene specific primers showed a specific band at ~2.0 kb with CC-4533 but not with fap265 mutant genomic DNA. (B) Equal amount of whole cell lysates from CC-4533 and fap265 mutant cells were separated on 12% SDS-PAGE; western blotting using antibodies against recombinant FAP265 detects a specific band at ~16.2 kDa in CC-4533 but not in fap265 lysates. Western blotting using antibodies against α-tubulin was used as loading control. (C) CC-4533 and fap265 cells were stained with antibodies against FAP265 (Red) and Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, SDS Page, Western Blot, Recombinant, Staining, Marker

    FAP265 localizes in the nucleus, (A, B) and at the cleavage furrow (C) in dividing CC-4533 cells. CC-4533 cells at different stages of cell division were fixed in methanol and stained with antibodies against α-tubulin (green) and FAP265 (red). Arrow heads mark nucleus and arrows mark cleavage furrow. Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: FAP265 localizes in the nucleus, (A, B) and at the cleavage furrow (C) in dividing CC-4533 cells. CC-4533 cells at different stages of cell division were fixed in methanol and stained with antibodies against α-tubulin (green) and FAP265 (red). Arrow heads mark nucleus and arrows mark cleavage furrow. Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: Staining

    Loss of FAP265 results in severe hatching defects and failure of daughter cells to assemble flagella in sporangium. (A) Bright field micrographs showing sporangium containing large number of daughter cells at steady state in fap265 mutant population compared with control cells. fap265 mutant cells are significantly bigger compared with wild type cells. Arrows mark flagella. Scale: 20μm (B) fap265 mutants and control cells in dividing stage were stained with antibodies against Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI. Scale: 10μm.
    Figure Legend Snippet: Loss of FAP265 results in severe hatching defects and failure of daughter cells to assemble flagella in sporangium. (A) Bright field micrographs showing sporangium containing large number of daughter cells at steady state in fap265 mutant population compared with control cells. fap265 mutant cells are significantly bigger compared with wild type cells. Arrows mark flagella. Scale: 20μm (B) fap265 mutants and control cells in dividing stage were stained with antibodies against Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI. Scale: 10μm.

    Techniques Used: Mutagenesis, Staining, Marker

    FAP265 localizes in flagella, basal bodies and cytoplasm in vegetative Chlamydomonas cells. (A) Coomassie gel showing lysates from whole cell and flagella of CC-4533 cells, separated on 12% SDS-PAGE. Western blotting using antibodies generated against recombinant FAP265 detects a specific band at ~16.2 kDa both in whole cell and flagellar fractions. Further, the blots were also probed with antibodies against NAB1 (Cytosolic marker). (B) Vegetative cells of wild type CC-4533 stained with antibodies against α-tubulin (green) and FAP265 (red). Arrows mark flagella and arrow head marks basal bodies. Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: FAP265 localizes in flagella, basal bodies and cytoplasm in vegetative Chlamydomonas cells. (A) Coomassie gel showing lysates from whole cell and flagella of CC-4533 cells, separated on 12% SDS-PAGE. Western blotting using antibodies generated against recombinant FAP265 detects a specific band at ~16.2 kDa both in whole cell and flagellar fractions. Further, the blots were also probed with antibodies against NAB1 (Cytosolic marker). (B) Vegetative cells of wild type CC-4533 stained with antibodies against α-tubulin (green) and FAP265 (red). Arrows mark flagella and arrow head marks basal bodies. Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: SDS Page, Western Blot, Generated, Recombinant, Marker, Staining

    Related Articles

    Centrifugation:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: Infected cells were clarified by centrifugation and RNA isolated by QIAamp MinElute Virus Spin Kit was frozen immediately. .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Amplification:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. A drawback of the two-enzyme systems is reduced efficiency during early rounds of RT-PCR amplification of low abundance targets when Taq Pol is used with MMLV RT , , , , , . .. This inhibition has some sequence specificity , , which presumably biases amplification and may compromise the measurement of differential gene expression levels and the reliability of internal and external quantification standards.

    Mutagenesis:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB). .. RNA was combined with primers and annealed in water at 70°C for 5 minutes followed by incubation on ice.

    Isolation:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: Infected cells were clarified by centrifugation and RNA isolated by QIAamp MinElute Virus Spin Kit was frozen immediately. .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Infection:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: Infected cells were clarified by centrifugation and RNA isolated by QIAamp MinElute Virus Spin Kit was frozen immediately. .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB). .. RNA was combined with primers and annealed in water at 70°C for 5 minutes followed by incubation on ice.

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. Use of 3173 Pol in RT-PCR We compared the first strand cDNA synthesis by 3173 Pol to that by MMLV RT using biological RNA templates. .. Production of cDNA was detected by two-step PCR amplification in which cDNA synthesis was primed by random, target-specific, oligo dT or no primers and detected by PCR with target-specific primers ( ).

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. The high stability of 3173 Pol in solution compared to MMLV RT also allows the formulation of a complete PyroScript RT-PCR master mix, which lacks only analyte-specific primers and target. ..

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. A drawback of the two-enzyme systems is reduced efficiency during early rounds of RT-PCR amplification of low abundance targets when Taq Pol is used with MMLV RT , , , , , . .. This inhibition has some sequence specificity , , which presumably biases amplification and may compromise the measurement of differential gene expression levels and the reliability of internal and external quantification standards.

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. The 3173 Pol was compared to MMLV RT for the detection of three shorter target sequences in common high-abundance reference genes using the two-step RT-PCR protocol. .. The amount of PCR product for all three transcripts appeared visibly greater in the 3173 Pol reactions, although we cannot rule out the contribution of residual thermostable 3173 Pol to the PCR reaction yield.

    Produced:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. The length distribution of the 3173 Pol cDNAs was visibly shorter than that produced by the MMLV RT, although a subset of the 3173 extension products appeared to be so large that they barely entered the gel. .. Incubation of the DNA primer:RNA template complex with the Taq Pol negative control resulted in a structure-dependent 5′-3′ exonuclease cleavage product that migrated at the dye front .

    Concentration Assay:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: The estimated MS2 RNA copy number was calculated from the determined concentration using an average molecular weight for an RNA base of 340 g mole−1 and the MS2 genome length of 3,569 nt. .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Incubation:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB). .. RNA was combined with primers and annealed in water at 70°C for 5 minutes followed by incubation on ice.

    other:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: The 3173 Pol and MMLV RT were both able to extend the primer to produce faint, nearly full-length products although the 3173 Pol product was detectably longer than that of MMLV RT.

    Activity Assay:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. As an additional test to compare the RT activity of the 3173 Pol to that of MMLV-RT on a complex RNA substrate, a primer specific to bases 714 to 690 of the negative sense RNA MS2 genome was 5′-fluorophore-labeled. .. The labeled cDNA primer was extended using extracted MS2 RNA as a template ( ).

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. Extension products from a 5′-fluorophore-labeled dT20 primer were resolved by denaturing polyacrylamide gel to further demonstrate RT activity and to assess the relative lengths of the extension products of the 3173 Pol and MMLV RT ( ). ..

    Molecular Weight:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: The estimated MS2 RNA copy number was calculated from the determined concentration using an average molecular weight for an RNA base of 340 g mole−1 and the MS2 genome length of 3,569 nt. .. Two-step RT-PCR reactions were performed using either 5 units 3173 Pol, exonuclease negative mutant or 200 Units MMLV RT (NEB).

    Sequencing:

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
    Article Snippet: .. Of the two longer target sequences tested, only the 821 bp beta-actin sequence (Lane C3) was reverse transcribed by the 3173 Pol and this synthesis appeared less efficient than that of MMLV RT. ..

    Plasmid Preparation:

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes
    Article Snippet: Klenow large fragment, Therminator™ II, M-MuLV (RNase H− ) reverse transcriptase, AMV reverse transcriptase, RNase inhibitor and λ-exonuclease were purchased from New England Biolabs. .. Plasmid HydGdCTD5 (5.176 kb,PCR template) was provided by Professor Peter Roach at Southampton University School of Chemistry.

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    New England Biolabs protoscript first strand cdna synthesis kit
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