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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Adaptor Ligation Mixture (E7370). protocols.io dx.doi.org/10.17504/protocols.io.cg6tzd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5®High-Fidelity DNA Polymerase, a novel thermostable DNA polymerase that possesses 3′ to 5′ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases.
Proper citation: New England Biolabs 2020. PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515): Amplification of bisulfite-converted, deaminated, or damaged DNA (Including FFPE DNA). protocols.io dx.doi.org/10.17504/protocols.io.7sdhna6 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol can be used to add As to the blunt-ends of DNA fragments that have been amplified using a high-fidelity polymerase (such as Q5® High Fidelity DNA Polymerase).
Proper citation: New England Biolabs 2015. A-Tailing with Taq Polymerase. protocols.io dx.doi.org/10.17504/protocols.io.crvv65 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Mixture for M0267 Taq with ThermoPol. protocols.io dx.doi.org/10.17504/protocols.io.cigubv Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The protocol described below will enable degradation of up to 20 pmol of a 25 nt ssDNA (~ 200 ng). In order to degrade larger amounts of ssDNA or ssDNAs longer than 25 nt, we recommend adding more enzyme instead of extending the reaction time. Users should note that ssDNAs longer than 25 nt may form secondary structures that hinder Thermolabile Exonuclease I activity.
Proper citation: New England Biolabs 2020. Protocol for removing ssDNA from dsDNA or RNA Samples (NEB #M0568). protocols.io dx.doi.org/10.17504/protocols.io.7ryhm7w Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. M0368 RNA and Primer mixture. protocols.io dx.doi.org/10.17504/protocols.io.chut6v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. M0348 Digestion Mixture. protocols.io dx.doi.org/10.17504/protocols.io.cr5v85 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. Mixture for M0492 Q5 2X Master Mix. protocols.io dx.doi.org/10.17504/protocols.io.cijucm Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the protocol for a large-scale Protein Expression Using BL21(DE3) Competent E. coli cells(C2527).
Proper citation: New England Biolabs 2014. Protein Expression Using BL21(DE3), Large Scale (C2527). protocols.io dx.doi.org/10.17504/protocols.io.cjrum5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. M0368 RT Mixture. protocols.io dx.doi.org/10.17504/protocols.io.chwt7d Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the blunting protocol for NEB PCR Cloning Kit (E1202)
Proper citation: New England Biolabs 2015. Blunting Protocol for NEB PCR Cloning Kit (E1202). protocols.io dx.doi.org/10.17504/protocols.io.crpv5m Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol details how to construct DNA 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 1.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 Standard Insert Libraries (370-420 bp) (NEB#E7120). protocols.io dx.doi.org/10.17504/protocols.io.bfuejnte Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the "quick" version of Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030). For the full protocol, please click here.
Proper citation: New England Biolabs 2016. Quick Protocol for Monarch® PCR & DNA Cleanup Kit (5 ?g) (NEB #T1030). protocols.io dx.doi.org/10.17504/protocols.io.ejxbcpn Copy
Authors: Isabel Gautreau
Group: New England Biolabs (NEB), Coronavirus Method Development Community
Summary: This protocol is about the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®).
Proper citation: Isabel Gautreau 2021. NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) (NEB #E7650S/L). protocols.io dx.doi.org/10.17504/protocols.io.bsy4nfyw Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the reaction for the "Ligation Protocol with T4 DNA Ligase"
Proper citation: New England Biolabs 2017. T4 DNA Ligase Reaction. protocols.io dx.doi.org/10.17504/protocols.io.irwcd7e Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. Single-temp DD Reaction. protocols.io dx.doi.org/10.17504/protocols.io.cjjukm Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Exo-CIP™ Rapid PCR Cleanup KitRapidly degrade residual PCR primers and dephosphorylate excess dNTPs after amplificationReaction complete in 4 minutesThermolabile formulation can be heat inactivated in 1 minute at 80°CPCR product can be used directly in downstream applicationsCompatible with commonly-used reaction buffers
Proper citation: New England Biolabs 2020. Protocol for Exo-CIP™ Rapid PCR Cleanup (#E1050). protocols.io dx.doi.org/10.17504/protocols.io.8yahxse Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol may be used with electrocompetent cells prepared by you according to this protocol.
Proper citation: New England Biolabs 2015. Electroporation Protocol. protocols.io dx.doi.org/10.17504/protocols.io.cruv6v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Mixture for M0530 Phusion PCR. protocols.io dx.doi.org/10.17504/protocols.io.cgztx5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the suggested protocol for use with λ DNA-Mono Cut Mix (N3019), фX174 DNA-HaeIII Digest (N3026),pBR322 DNA-BstNI Digest (N3031), pBR322 DNA-MspI Digest (N3032), 2-Log DNA Ladder (0.1-10.0 kb) (N3200), 100 bp DNA Ladder (N3231), 1 kb DNA Ladder (N3232), Low Molecular Weight DNA Ladder (N3233), and 50 bp DNA Ladder (N3236)
Proper citation: New England Biolabs 2015. Suggested protocol for loading a DNA Ladder/marker. protocols.io dx.doi.org/10.17504/protocols.io.cq4vyv Copy
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