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polya polymerase buffer  (New England Biolabs)


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    New England Biolabs polya polymerase buffer
    Polya Polymerase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/polya+buffer/pm39198865-219-15-18
    Average 86 stars, based on 1 article reviews
    polya polymerase buffer - by Bioz Stars, 2026-10
    86/100 stars

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    Incubation:

    Article Title: NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs
    Article Snippet: For NERD-seq, the standard protocol was further modified as follows: 1.5ug total RNA was separated into two fractions (short and long RNA) using the Invitrogen MirVana kit (AM1561) with a modified protocol as described before in our short RNA-seq approach for illumina ( – ). .. 90uL of the short RNA elution fraction was polyadenylated using 12uL 10x polyA polymerase buffer (NEB, B0276), 12uL 10mM ATP (NEB, B0756A), 6uL polyA polymerase (NEB, M0276) and incubated at 37C for 30min. .. The polyadenylated short fraction and the long fraction were combined and purified and concentrated using the RNeasy MinElute kit (Qiagen, 74204).

    Article Title: NERD-seq: a novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs.
    Article Snippet: In both the standard and the NERD-seq protocol, ligations were performed for 15 min. For NERD-seq, the standard protocol was further modified as follows: 1.5 μg total RNA was PNK digested using 50U PNK (NEB, M0201) and 10 μL 10X PNK buffer for 1 h at 37 °C, then directly separated into two fractions (short and long RNA) using the Invitrogen MirVana kit (AM1561) with a modified protocol as described before in our short RNA-seq approach for Illumina [48, 50, 105, 106]. .. Ninety microliters of the short RNA elution fraction was polyadenylated using 12 μL 10 × polyA polymerase buffer (NEB, B0276), 12 μL 10 mM ATP (NEB, B0756A), 6 μL polyA polymerase (NEB, M0276), and incubated at 37 °C for 30 min. .. The polyadenylated short fraction and the long fraction were combined, purified, and concentrated using the RNeasy MinElute kit (Qiagen, 74,204) to a final volume of 12 μL of RNA that was adaptor ligated using 4 μL NEBNext Quick ligation buffer (B6058), 0.66 μL RNA CS (Nanopore), 1.3 μL RTA adaptor (Nanopore), 2 μL 2,000,000U T4 DNA ligase (NEB, M0202).



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    Plot showing the sequencing coverage (green), location of the 3’ ends of reads (orange) and splicing patterns (purple lines connecting splice donors and acceptors) for two Wiskott-Aldrich Syndrome vectors sequenced by Nanopore direct RNA technology (A) WAS LV5 sequencing data (contains a single A to T mutation in the potential cryptic polyA site). (B) Reference sequence of the Wiskott-Aldrich Syndrome vector, with various features annotated.

    Journal: bioRxiv

    Article Title: An optimised protocol for quality control of gene therapy vectors using Nanopore direct RNA sequencing

    doi: 10.1101/2023.12.03.569756

    Figure Lengend Snippet: Plot showing the sequencing coverage (green), location of the 3’ ends of reads (orange) and splicing patterns (purple lines connecting splice donors and acceptors) for two Wiskott-Aldrich Syndrome vectors sequenced by Nanopore direct RNA technology (A) WAS LV5 sequencing data (contains a single A to T mutation in the potential cryptic polyA site). (B) Reference sequence of the Wiskott-Aldrich Syndrome vector, with various features annotated.

    Article Snippet: Artificial polyadenylation was carried out based on Crabtree et al. ( ) Reactions containing 15 μL of RNA (500 ng), 2 μL of polyA buffer, 2 μL of 10 mM ATP (NEB P0756S), 1 μL of E. coli Poly(A) Polymerase (NEB M0276S) and 0.5 μL of Murine RNase Inhibitor (NEB M0314S) were incubated for 30 minutes at 37°C, 20 minutes at 65°C and then 5 minutes at 98°C before being placed on ice.

    Techniques: Sequencing, Mutagenesis, Plasmid Preparation