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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.
Proper citation: New England Biolabs 2018. Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765). protocols.io dx.doi.org/10.17504/protocols.io.tunenve Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II FS DNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows.
Proper citation: New England Biolabs 2021. E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol to use with Inputs ≥ 100 ng (Chapter 2). protocols.io dx.doi.org/10.17504/protocols.io.nwxdffn Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal Alkaline Phosphatase (CIP - M0290)
Proper citation: New England Biolabs 2014. Dephosphorylation using CIP in Restriction Enzyme Reaction (M0290). protocols.io dx.doi.org/10.17504/protocols.io.cdhs35 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol is for PCR with Q5® High-Fidelity 2X Master Mix (M0492)
Proper citation: New England Biolabs 2014. PCR with Q5® High-Fidelity 2X Master Mix (M0492). protocols.io dx.doi.org/10.17504/protocols.io.ciiucd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for non-radioactive phosphorylation with T4 PNK.
Proper citation: New England Biolabs 2015. Non-radioactive phosphorylation with T4 PNK. protocols.io dx.doi.org/10.17504/protocols.io.cpdvi5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Both the MBP-tag and TEV Protease are polyhistidine-tagged for easy removal from the TEV Protease reaction. Loading the digest onto NEBExpress Ni Resin (NEB #S1428) sequesters both the MBP-tag and TEV Protease, thereby isolating the protein of interest in the column flow through.
The binding capacity of NEBExpress Ni Resin is ≥ 10 mg/ml. The binding capacity will vary depending on the target protein, binding conditions and the accessibility of the His-tag.
It is recommended to estimate the amount of the His-tagged MBP released by TEV Protease cleavage by first analyzing a sample by SDS-PAGE and comparing its band size and intensity to that of the provided MBP6 protein control.
Proper citation: New England Biolabs 2020. Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201). protocols.io dx.doi.org/10.17504/protocols.io.bfebjjan Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for non-radioactive phosphorylation with T4 PNK 3' phosphatase minus.
Proper citation: New England Biolabs 2015. Non-radioactive phosphorylation with T4 PNK 3' phosphatase minus. protocols.io dx.doi.org/10.17504/protocols.io.cp2vqd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the correct protocol if you are using the C3019I cells. If you are using the C3019H cells, please refer to this protocol.
Proper citation: New England Biolabs 2018. High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019I). protocols.io dx.doi.org/10.17504/protocols.io.nkvdcw6 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. E1202 Blunting Mixture. protocols.io dx.doi.org/10.17504/protocols.io.crqv5v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the quick version of the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). For the full protocol, please click here.
Proper citation: New England Biolabs 2016. Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010). protocols.io dx.doi.org/10.17504/protocols.io.ejybcpw Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Radioactive Labeling with T4 Polynucleotide Kinase 3' phosphatase minus
Proper citation: New England Biolabs 2015. Radioactive Labeling with T4 PNK 3' phosphatase minus. protocols.io dx.doi.org/10.17504/protocols.io.cp9vr5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the protocol for the Gibson Assembly using the Gibson Assembly® Master Mix (E2611).
Proper citation: New England Biolabs 2015. Gibson Assembly® Master Mix – Assembly (E2611). protocols.io dx.doi.org/10.17504/protocols.io.cjxupm Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Capped RNA E2050 Mixture. protocols.io dx.doi.org/10.17504/protocols.io.crzv75 Copy
Authors: New England BioLabs, Inc.
Group: New England Biolabs (NEB)
Summary: Cas9 nuclease may be used in vivo to create targeted genome modifications. There are several ways in which to introduce Cas9-guide RNA complexes into cells. Here we present a method for the transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX. This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.
Proper citation: New England BioLabs, Inc. 2016. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX. protocols.io dx.doi.org/10.17504/protocols.io.frjbm4n Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the correct protocol if you are using the C3019H cells. If you are using the C3019I cells, please refer to this protocol.
Proper citation: New England Biolabs 2015. High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019H). protocols.io dx.doi.org/10.17504/protocols.io.chct2v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: OneTaq DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons regardless of GC content.
Proper citation: New England Biolabs 2015. PCR Protocol for OneTaq® DNA Polymerase (M0480). protocols.io dx.doi.org/10.17504/protocols.io.cq8vzv Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2018. Restriction Digest Reaction. protocols.io dx.doi.org/10.17504/protocols.io.iszcef6 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)
Proper citation: New England Biolabs 2015. Large (Klenow) Fragment Blunting (M0210). protocols.io dx.doi.org/10.17504/protocols.io.cpgvjv Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030)
Proper citation: New England Biolabs 2020. Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 ?g) (NEB #T1030). protocols.io dx.doi.org/10.17504/protocols.io.bp9dmr26 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol details how to construct Large Insert libraries from start to finish using NEBNext reagents. The corresponding NEB manual is here: https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf and this protocol relates to section 2.OverviewThe Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymatically modified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC). Figure 1 is an overview of the EM-seq workflow. Firstly, a library is made by ligating EM-seq adaptor to sheared end repaired/dA-tailed genomic DNA. This is followed by two sets of enzymatic conversion steps to differentiate cytosines from 5mC and 5hmC. Finally, libraries are PCR amplified before sequencing.Figure 2 shows a comparison of the sodium bisulfite and EM-seq methods. Sodium bisulfite treatment of DNA results in the deamination of cytosines into uracils, however the modified forms of cytosine (5mC and 5hmC) are not deaminated. Therefore, the preference of bisulfite to chemically deaminate cytosines enables the methylation status of cytosines to be determined. When bisulfite treated DNA is PCR amplified, uracils are replaced by thymines and the 5mC/5hmC are replaced by cytosines. Once sequenced, unmethylated cytosines are represented by thymines and 5mC and 5hmC are represented by cytosines. By comparing sequences to non-converted genomes the appropriate methylation status can be assessed.Enzymatic Methyl-seq is a two step enzymatic conversion process to detect modified cytosines. The first step uses TET2 and an oxidation enhancer to protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC through a cascade reaction into 5-carboxycytosine [5-methylcytosine (5mC) Þ5-hydroxymethylcytosine (5hmC) Þ 5-formylcytosine (5fC) Þ 5- carboxycytosine (5caC)]. This protects 5mC and 5hmC from deamination. 5hmC can also be protected from deamination by glucosylation to form 5ghmc using the oxidation enhancer. The second enzymatic step uses APOBEC to deaminate C but does not convert 5caC and 5ghmC. The resulting converted sequence can be analyzed like bisulfite-treated DNA. Typical aligners used to analyze data include but are not limited to Bismark and BWAMeth.The workflow described in the NEBNext Enzymatic Methyl-seq Kit is user-friendly and enables methylation detection from inputs ranging between 10 ng–200 ng. EM-seq converted DNA is more intact than bisulfite-converted DNA, resulting in libraries with longer sequencing reads, reduced GC bias and more even genome coverage. Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction of indexed libraries and sequenced on an Illumina sequencing platform. For larger volume requirements, customized and bulk packaging is available by purchasing through the Custom Solutions department at NEB. Please contact [email protected] for further information.
Proper citation: New England Biolabs 2020. Protocol for Use with Large Insert Libraries (470–520 bp) (NEB #E7120). protocols.io dx.doi.org/10.17504/protocols.io.bfu2jnye Copy
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