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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 2015. T4 DNA Ligase Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.cf7trm Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. PNGase F Mixture 1. protocols.io https://dx.doi.org/10.17504/protocols.io.cqgvtv Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Standard RNA Synthesis Mixture for E2050. protocols.io https://dx.doi.org/10.17504/protocols.io.cr3v8m Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. BioBrick E0546 Upstream Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.cpivkd Copy
Authors: New England Biolabs
Group: Grupo de Estudos de Leveduras Cervejeiras
Proper citation: New England Biolabs 2015. SOB Media. protocols.io https://dx.doi.org/10.17504/protocols.io.crrv55 Copy
Authors: New England Biolabs
Summary: This protocol defines methods for the Q5® Site-Directed Mutagenesis Kit without competent cells.
Proper citation: New England Biolabs 2020. Q5® Site-Directed Mutagenesis (E0552). protocols.io https://dx.doi.org/10.17504/protocols.io.beurjev6 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is to be performed with the Quick Ligation Reaction Buffer. Please see the NEB website for more information.
Proper citation: New England Biolabs 2014. Quick Ligation Protocol (M2200). protocols.io https://dx.doi.org/10.17504/protocols.io.cdgs3v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. M0368 RT Mixture. protocols.io https://dx.doi.org/10.17504/protocols.io.chwt7d 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 T4 DNA Polymerase.
Proper citation: New England Biolabs 2015. Blunting Protocol (M0203). protocols.io https://dx.doi.org/10.17504/protocols.io.cgwtxd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The BioBrick® Assembly Kit was developed in partnership with Ginkgo BioWorks. What follows is an abbreviated set of protocols for the use of the BioBrick® Assembly Kit (to assemble an Upstream Part with a Downstream Part into a Destination Plasmid). For more details and for technical questions, please see here.
Proper citation: New England Biolabs 2015. Digestion for BioBrick Assembly Kit (E0546). protocols.io https://dx.doi.org/10.17504/protocols.io.cphvj5 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 https://dx.doi.org/10.17504/protocols.io.crpv5m Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
Proper citation: New England Biolabs 2015. Dephosphorylation of 5´-ends of DNA using rSAP (M0371). protocols.io https://dx.doi.org/10.17504/protocols.io.crkv4v 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 https://dx.doi.org/10.17504/protocols.io.bfuejnte Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. Digestion Mixture for M0302. protocols.io https://dx.doi.org/10.17504/protocols.io.cqrvv5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is a protocol for a typical DNase I Reaction, using the M0303 RNase-free DNase I.
Proper citation: New England Biolabs 2015. A Typical DNase I Reaction (M0303). protocols.io https://dx.doi.org/10.17504/protocols.io.cqevtd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This electroporation protocol is for use with the NEB Turbo Electrocompetent E. coli cells (C2986). These cells are suitable for high efficiency electroporation and rapid colony growth; they are ideal for DNA library constructions and all cloning purposes.
Proper citation: New England Biolabs 2015. Electroporation Protocol (C2986). protocols.io https://dx.doi.org/10.17504/protocols.io.crgv3v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is a generic PNGase F protocol with non-denaturing reaction conditions. It is appropriate for both P0704 and P0708. Typical reaction conditions are below.
Proper citation: New England Biolabs 2015. PNGase F Protocol, Non-Denaturing Conditions. protocols.io https://dx.doi.org/10.17504/protocols.io.cqivud Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. BioBrick E0546 Destination Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.cpmvk5 Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. NEB 3X SDS Sample Loading Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.crcv2v Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2014. FirstStrand with RT Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.cjvun5 Copy
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