NEB
A scientific update
Issue I • 2022
SCIENCE
INNOVATION
Minding your caps and tails – considerations for functional mRNA synthesis
Feature Article
1
– Yield 1.8 mg of Cap-1mRNA per Kit! – Decrease dsRNA byproduct
(m)RNA Synthesis/IVT
2
Modern DNA Assembly reformulated – NEBridge Ligase for GGA – Competent Cells at Special prices
Clone with confidence
3
– Lyophilized Luna RT-qPCR Kit now available – Highest Fidelity Multiplexing RT-PCR
Lighting the way
4
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NGS Library Prep
5
USA New England Biolabs, Inc. Telephone: (978) 927-5054 Toll Free: (U.S. Orders) 1-800-632-5227 Toll Free: (U.S. Tech) 1-800-632-7799 www.neb.com
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DNA Ladders
6
Lithuania: Nanodiagnostika, Ltd. Tel: (+370) 525 052 44 lina@nanodiagnostika.lt www.nanodiagnostika.lt
by Breton Hornblower, Ph.D., G. Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New England Biolabs, Inc.
Learn more about HiScribe T7 mRNA Kit with CleanCap Reagent AG
Back to top
Applications of synthetic mRNA have grown and become considerably diversified in recent years. Examples include the generation of pluripotent stem cells (1-3), vaccines and therapeutics (4-6), and CRISPR/Cas9 genome editing applications (7-9). The basic requirements for a functional mRNA – a 7-methylguanylate cap at the 5´ end and a poly(A) tail at the 3´ end – must be added in order to obtain efficient translation in eukaryotic cells. Additional considerations can include the incorporation of internal modified bases, modified cap structures and polyadenylation strategies. Strategies for in vitro synthesis of mRNA vary according to the desired scale of synthesis.
A nascent mRNA, synthesized in the nucleus, undergoes modifications before it can be translated into proteins in the cytoplasm. For a mRNA to be functional, it requires modified 5´ and 3´ ends and a coding region (i.e., an open reading frame (ORF) encoding the protein of interest) flanked by the untranslated regions (UTRs). The nascent mRNA (pre-mRNA) undergoes two significant modifications in addition to splicing. During synthesis, a 7-methylguanylate structure, also known as a “cap”, is added to the 5´ end of the pre-mRNA, via 5´ → 5´ triphosphate linkage. This cap protects the mature mRNA from degradation, and also serves a role in nuclear export and efficient translation. The second modification occurs post-transcriptionally at the 3´ end of the nascent RNA molecule, and is characterized by addition of approximately 200 adenylate nucleotides (poly(A) tail). The addition of the poly(A) tail confers stability to the mRNA, aids in the export of the mRNA to the cytosol, and is involved in the formation of a translation-competent ribonucleoprotein (RNP), together with the 5´ cap structure. The mature mRNA forms a circular structure (closed-loop) by bridging the cap to the poly(A) tail via the cap-binding protein eIF4E (eukaryotic initiation factor 4E) and the poly(A)-binding protein, both of which interact with eIF4G (eukaryotic initiation factor 4G) (10). RNA can be efficiently synthesized in vitro (by in vitro transcription, IVT) with prokaryotic phage polymerases, such as T7, T3 and SP6. The cap and poly(A) tail structures characteristic of mature mRNA can be added during or after the synthesis by enzymatic reactions with capping enzymes and Poly(A) Polymerase (NEB #M0276), respectively. There are several factors to consider when planning for IVT-mRNA synthesis that will influence the ease-of-experimental setup and yield of the final mRNA product.
In vitro transcription
There are two options for the in vitro transcription (IVT) reaction depending on the capping strategy chosen: standard synthesis with enzyme-based capping following the transcription reaction (post-transcriptional capping) or incorporation of a cap analog during transcription (co-transcriptional capping) (Figure 1). Method selection will depend on the scale of mRNA synthesis required and number of templates to be transcribed.
Figure 1: In vitro transcription options based upon capping strategy
Standard RNA synthesis reactions produce the highest yield of RNA transcript (typically ≥100 μg per 20 μl in a 1 hr reaction using the HiScribe Quick T7 High Yield RNA Synthesis Kit, NEB #E2050S). Transcription reactions are highly scalable. Following transcription, the RNA is treated with DNase I (NEB #M0303) to remove the DNA template, and purified using an appropriate column, kit or magnetic beads, prior to capping. This method produces high yields of RNA with 5´-triphosphate termini that must be converted to cap structures. In the absence of template-encoded poly(A) tails, transcripts produced using this method bear 3´ termini that also must be polyadenylated in a separate enzymatic step, as described below in “Post-transcriptional capping and Cap-1 methylation”.
Transcription for enzyme-based capping (post-transcriptional capping)
In co-transcriptional capping, a cap analog is introduced into the transcription reaction, along with the four standard nucleotide triphosphates, in an optimized ratio of cap analog to GTP 4:1. This allows initiation of the transcript with the cap structure in a large proportion of the synthesized RNA molecules. This approach produces a mixture of transcripts, of which ~80% are capped, and the remainder have 5´-triphosphate ends. Decreased overall yield of RNA products results from the lower concentration of GTP in the reaction. There are several cap analogs used in co-transcriptional RNA capping (3). The most common are the standard 7-methyl guanosine (m7G) cap analog and anti-reverse cap analog (ARCA), also known as 3´ O-me 7-meGpppG cap analog. ARCA is methylated at the 3´ position of the m7G, preventing RNA elongation by phosphodiester bond formation at this position. Thus, transcripts synthesized using ARCA contain 5´-m7G cap structures in the correct orientation, with the 7-methylated G as the terminal residue. In contrast, the m7G cap analog can be incorporated in either the correct or the reverse orientation. HiScribe T7 ARCA mRNA Synthesis kits (NEB #E2060 and #E2065) contain reagents, including an optimized mix of ARCA and NTPs, for streamlined reaction setup for synthesis of co-transcriptionally capped RNAs.
Transcription with dinucleotide co-transcriptional capping
The use of CleanCap reagent AG results in significant advantages over traditional dinucleotide co-transcriptional capping. CleanCap Reagent AG is a trinucleotide with a 5´-m7G joined by a 5´ → 5´ triphosphate linkage to an AG sequence. The adenine has a methyl group on the 2´-O position. The incorporation of this trinucleotide in the beginning of a transcript results in a Cap-1 structure. In order to use CleanCap Reagent AG in an in vitro transcription reaction the template must contain an AG in place of a GG following the T7 promoter in the initiation sequence. Unlike traditional co-transcriptional capping, reduction of GTP concentration is not required and therefore yield is higher and high capping effiencies, >95%, are achieved.
Transcription with CleanCap Reagent AG co-transcriptional capping
RNA synthesis can be carried out with a mixture of modified nucleotides in place of the regular mixture of A, G, C and U triphosphates. For expression applications, the modified nucleotides of choice are the naturally occurring 5´-methylcytidine and/or pseudouridine in the place of C and U, respectively. These have been demonstrated to confer desirable properties to the mRNA, such as increased mRNA stability, increased translation, and reduced immune response in the key applications of protein replacement and stem-cell differentiation (1). It is important to note that nucleotide choice can influence the overall yield of mRNA synthesis reactions. Fully substituted RNA synthesis can be achieved using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080), HiScribe T7 High-Yield RNA Synthesis Kit (NEB #E2040) or HiScribe SP6 RNA Synthesis Kit (NEB #E2070) in conjunction with NTPs with the desired modification. Transcripts made with complete replacement of one or more nucleotides may be post-transcriptionally capped (see next section), or may be co-transcriptionally capped by including CleanCap Reagent AG, ARCA or another cap analog, as described previously. If partial replacement of nucleotides is desired, the HiScribe T7 ARCA mRNA Synthesis Kits (NEB #E2060 and #E2065), may be used with added modified NTPs, to produce co-transcriptionally capped mRNAs, as described above. Alternatively, the HiScribe T7 Quick RNA Synthesis Kit (NEB #E2050) may be used to prepare transcripts for post-transcriptional capping.
Transcription with complete substitution with modified nucleotides
Post-transcriptional capping is often performed using the mRNA capping system from Vaccinia virus. This enzyme complex converts the 5´-triphosphate ends of in vitro transcripts to m7G-cap (Cap-0) required for efficient protein translation in eukaryotes. The Vaccinia Capping System (NEB #M2080) comprises three enzymatic activities (RNA triphosphatase, guanylyltransferase, guanine N7-methyltransferase) that are necessary for the formation of the complete Cap-0 structure, m7Gppp5´N, using GTP and the methyl donor S-adenosylmethionine (SAM). As an added option, the inclusion of the mRNA Cap 2´ O-Methyltransferase (NEB #M0366) in the same reaction results in formation of the Cap-1 structure (m7Gppp5´Nm), a natural modification in many eukaryotic mRNAs responsible for evading cellular innate immune response against foreign RNA. This enzyme-based capping approach results in a high proportion of capped message, and it is easily scalable. The resulting capped RNA can be further modified by poly(A) addition before final purification.
Post-transcriptional capping and Cap-1 methylation
The poly(A) tail confers stability to the mRNA and enhances translation efficiency. The poly(A) tail can be encoded in the DNA template by using an appropriately tailed PCR primer, or it can be added to the RNA by enzymatic treatment with E. coli Poly(A) Polymerase (NEB #M0276). The length of the added tail can be adjusted by titrating the Poly(A) Polymerase in the reaction. For mRNA synthesis from templates with encoded poly(A) tails, the HiScribe T7 ARCA mRNA Synthesis Kit (NEB #E2065) provides an optimized formulation for co-transcriptionally capped transcripts. In summary, when choosing the right workflow for your functional mRNA synthesis needs, you must balance your experimental requirements for the mRNA (e.g., internal modifed nucleotides) with scalability (i.e., ease-of-reaction setup vs. yield of final product). Products from NEB are available for each step of the RNA synthesis workflow. GMP-grade* reagents suitable for the large scale manufacture of therapeutics mRNA are available through our Customized Solution Group.
A-tailing using E. coli Poly(A) Polymerase
Enzyme-based capping (top) is performed after in vitro transcription using 5´-triphosphate RNA, GTP, and S-adenosyl- methionine (SAM). Cap-0 mRNA can be converted to Cap-1 mRNA using mRNA cap 2´-O-methyltransferase (MTase) and SAM in a subsequent or concurrent reaction. The methyl group transferred by the MTase to the 2´-O of the first nucleotide of the transcript is indicated in red. Conversion of ~100% of 5´-triphosphorylated transcripts to capped mRNA is routinely achievable using enzyme-based capping. Co-transcriptional capping (bottom) uses an mRNA cap analog, shown in yellow, in the transcription reaction. For ARCA (anti-reverse cap analog) (left),the cap analog is incorporated as the first nucleotide of the transcript. ARCA contains an additional 3´-O-methyl group on the 7-methylguanosine to ensure incorporation in the correct orientation. The 3´-O-methyl modification does not occur in natural mRNA caps. Compared to reactions not containing cap analog, transcription yields are lower. ARCA- capped mRNA can be converted to cap 1 mRNA using mRNA cap 2´-O-MTase and SAM in a subsequent reaction. CleanCap Reagent AG (right) uses a trinucleotide cap analog that requires a modified template initiation sequence. A natural Cap-1 structure is accomplished in a co-transcriptional reaction.
"GMP-grade" is a branding term NEB uses to describe reagents manufactured at NEB’s Rowley facility. The Rowley facility was designed to manufacture reagents under more rigorous infrastructure and process controls to achieve more stringent product specifications and customer requirements. Reagents manufactured at NEB’s Rowley facility are manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards. However, at this time, NEB does not manufacture or sell products known as Active Pharmaceutical Ingredients (APIs), nor does NEB manufacture its products in compliance with all of the Current Good Manufacturing Practice regulations. References: 1. Warren, L., et al. (2010) Cell Stem Cell, 7, 618-630. 2. Angel, M. and Yanik, M.F. (2010) PLoS One, 5:e11756. 3. Yakubov, E., et al. (2010) Biochem. Biophys. Res. Commun. 394, 189. 4. Haas, E.J., et al. (2021) Lancet, 397, 1819-1829. 5. Thompson, M.G., et al. (2021) N. Engl. J. Med. 384, 403-416. 6. Ramaswamy, S., et al. (2017) Proc. Natl. Acad. Sci. USA, 114, E1941-E1950. 7. Ma, Y., et al. (2014) PLoS One, 9:e89413. 8. Ota, S., et al. (2014) Genes Cells, 19, 555-564. 9. Bassett, A. R., et al. (2013) Cell Rep. 4, 220–228. 10. Wells, S.E., et al. (1998) Molecular Cell 2, 135–140.
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Extract from Article:
The HiScribe T7 mRNA Kit with CleanCap Reagent AG utilizes an optimized RNA synthesis formulation and trinucleotide cap analog technology to co-transcriptionally cap mRNAs containing a natural Cap-1 structure in a single simplified reaction without compromising RNA yield. Using a DNA template with a T7 promoter sequence followed by an AG initiation sequence and an encoded poly(A) tail, mRNAs can be transcribed with a 5´-m7G Cap-1 structure that is polyadenylated, translationally competent and able to evade the cellular innate immune response.
Effectively purify total RNA of all sizes, including small RNA (<200 nt)
Validated for viral RNA extraction from clinically-relevant samples (automatable on the QIAcube® and KingFisher™ Flex)
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Reagents, and adaptors and primers (12- and 96-index) sold separately
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Restriction enzymes from NEB — same high performance, now with BSA-free reaction buffer
To address the growing need for comparable performance using BSA-free reagents, we have begun switching our current BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). These buffers have been rigorously tested, and there is no difference in performance when using either system. This switch started in April 2021, but may take up to 6 months to complete. Over this time, you may receive product with BSA- or rAlbumin-containing buffer — either will work for your reactions.
New tools for your (m)RNA Synthesis (IVT)
New Products: HiScribe T7 mRNA Kit with CleanCap Reagent AG
Suitable for full or partial modified nucleotide substitutions
Generate high yields of mRNA, up to 1.8 mg per kit
Evade immune response with natural Cap-1 structure
Synthesize and cap mRNA in a single reaction
Comparison of RNA Yields from in vitro Reagent AG Transcription Reactions with no cap analog, ARCA, or CleanCap Reagent AG
All reactions were performed with 5 mM CTP, 5 mM UTP and 6 mM ATP. Standard IVT reactions included 5 mM GTP and no cap analog. ARCA reactions contained a 4:1 ratio of ARCA:GTP (4mM:1mM). IVT with CleanCap Reagent AG contained 5 mM GTP and 4 mM CleanCap Reagent AG and was performed as described (Standard mRNA Synthesis, HiScribe T7 mRNA Kit with CleanCap Reagent AG). Reactions were incubated for 2 hours at 37°C, purified and quantified by NanoDrop.
Product
NEB #
Size
HiScribe T7 mRNA Kit with CleanCap Reagent AG
E2080S
20 rxns
Flexibility – enables incorporation of cap analogs, radiolabeled and modified nucleotides
High Yield – up to 180 μg of RNA from a standard 20 μl reaction
Streamlined format & Quick Workflows
HiScribe T7 High Yield RNA Synthesis Kit
E2040S
50 rxns
The HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040) delivers robust high yield RNA synthesis (up to 180 µg/reaction) for a wide range of template sizes. Flexible protocols ensure that performance is maintained even under demanding conditions, such as extended reaction time using very low amounts of template. Protocols are included for partial or complete incorporation of modified or labeled nucleotides in the transcript body, and cap analogs at the RNA 5' end. The HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050) utilizes a master mix format, allowing for faster reaction setup. DNase I and lithium chloride are included for DNA template removal and quick RNA purification.
Also available: HiScribe™ T7 (Quick) High Yield RNA Synthesis Kit
HiScribe T7 Quick High Yield RNA Synthesis Kit
E2050S
HiScribe SP6 RNA Synthesis Kit
E2070S
Decreased unwanted immunogenicity from RNA synthesized at higher temperature due to reduced dsRNA by-product formation
Increased co-transcriptional capping efficiency with cap analogs
Active from 37-56°C, optimal incubation temperature is 50-52°C
Hi-T7 RNA Polymerase (High Concentration)
M0470T
50.000 units
Hi-T7 RNA Polymerase
M0658S
5.000 units
Hi-T7 RNA Polymerase is an engineered DNA-dependent RNA polymerase that is highly specific for T7 phage promoters, designed for in vitro transcription of RNA at higher temperatures and recommended for experienced users interested in building and optimizing their own in vitro transcription reactions.
Hi-T7 RNA Polymerase for reduced dsRNA by-product formation
Immunoblot using an anti-dsRNA antibody (J2) shows presence of dsRNA by-products in the IVT reactions for both T7 and Hi-T7 RNA Polymerases when IVT is performed at 37°c. HPLC purification of the IVT RNA eliminates dsRNA by-products. dsRNA by-products are reduced when IVT is performed at 50°c (or higher temperatures) with Hi-T7.
Get caught up on key mRNA discoveries with our interactive timeline, featuring a selection of publications and resources from the last seven decades.
Explore our interactive timeline of mRNA discoveries
Learn more about mRNA
• RNA Technical Guide – Find in depth information on tools designed to streamline your RNA workflows • RNA Synthesis Brochure – Learn more about NEB's products for RNA synthesis, which range from template generation to poly(A) tailing • GMP-Grade* Reagents for RNA Synthesis Brochure – Learn about the benefits of GMP-grade materials available from NEB, and how they can be used in your mRNA synthesis workflow
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"GMP-grade" is a branding term NEB uses to describe reagents manufactured at NEB’s Rowley facility. The Rowley facility was designed to manufacture reagents under more rigorous infrastructure and process controls to achieve more stringent product specifications and customer requirements. Reagents manufactured at NEB’s Rowley facility are manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards. However, at this time, NEB does not manufacture or sell products known as Active Pharmaceutical Ingredients (APIs), nor does NEB manufacture its products in compliance with all of the Current Good Manufacturing Practice regulations.
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"Standard-size" cloning and larger gene assembly products, up to 11 fragments
Seamless and ordered HiFi cloning in as little as 15 minutes
NEBuilder HiFi DNA Assembly Master Mix
E2621S/ L/X
10 /50 / 250 rxns
Assembling DNA fragments is a key part of both synthetic biology techniques and cloning. NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those with 5´- and 3´-end mismatches. This flexible kit enables simple and fast seamless cloning utilizing a proprietary high-fidelity polymerase. Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning.
NEBuilder HiFi DNA Assembly – reformulated for improved performance
Not your average DNA assembly reagent
NEBuilder HiFi DNA Assembly Cloning Kit
E5520S
10 rxns
NEBuilder HiFi DNA Assembly Bundle for Large Fragments
E2623S
Use for seamless cloning – no scar remains following assembly
Use with NEB Type IIS restriction enzymes
Optimized for efficient and accurate Golden Gate Assembly
NEBridge Ligase Master Mix
M1100S
Offering flexibility and convenience for users, NEBridge Ligase Master Mix performs high efficiency and high-fidelity Golden Gate Assembly with a broad range of NEB Type IIS restriction enzymes. NEBridge Ligase Master Mix is a 3X master mix for Golden Gate Assembly. Designed for use with NEB Type IIS restriction enzymes, this master mix contains T4 DNA Ligase in an optimized reaction buffer with a proprietary ligation enhancer. Users only need to choose their preferred NEB Type IIS restriction enzyme and add DNA substrates to be assembled. Low complexity single-fragment insertions, as well as moderate complexity (3–6 fragment) and high complexity (7–25+ fragment) assemblies, are all supported with this optimized reagent and accompanying protocols.
NEBridge™ Ligase Master Mix
Ideal for ordered assembly of multiple fragments (2-25+) in a single reaction
Can also be used for cloning single inserts and library construction
Design primers with our free tool available at GoldenGate.neb.com
Bulk formats and custom packaging are available
Choose from a variety of convenient formats, including single-use tubes
Outgrowth medium and control plasmid are included
All strains are free of animal products and are T1 phage resistant
Compatible with NEBuilder HiFi DNA Assembly and NEBridge Golden Gate Assembly reactions, as well as ligation reactions. No dilution required!
High transformation efficiencies
NEB’s growing line of competent cells includes several popular strains for cloning. Our cloning strains include derivatives of the industry standards, DH5α and DH10B. NEB Turbo is unique to NEB and allows colony growth after 6.5 hours. NEB’s dam–/dcm– strain enables isolation of plasmids free of Dam and Dcm methylation. NEB Stable is recommended in all difficult cloning experiments. Our cells are all extensively tested for phage resistance, antibiotic resistance and sensitivity, blue/white screening and transformation efficiency. High efficiency, 5 minute transformation and electroporation protocols are provided, when applicable.
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Transformation efficiencies were compared using manufacturers’ recommended protocols. Values shown are the average of triplicate experiments.
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Eliminate cold chain shipping requirements
Store at room temperature for up to 2 years prior to rehydration
Simply add nuclease-free water for rapid rehydration
LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG
L4001S
120 rxns
Nuclease-free Water
B1500S/L
25/100 ml
With the point-of-care market becoming more focused on the development of robust, accurate and cost-effective diagnostic tests for use outside of traditional hospital and laboratory settings, there is a growing need for reagents that can withstand ambient shipping and storage. Lyophilization is the preferred solution and is a well-established technology across a number of industries. Bringing together expertise in enzyme development, manufacturing and lyophilization, NEB and Fluorogenics Limited have created shelf-stable, lyophilized products that do not sacrifice the high-performance qualities of their liquid counterparts. The first of these products includes a mixture of enzymes and inhibitors to enable robust detection of RNA via hydrolysis-probe-based RT-qPCR. LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) contains the same versatile features and strong performance as the liquid version: Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019). Performance in multiplexing applications has been optimized, with sensitive, linear detection of up to 5 targets across a range of inputs. The stable cake can be resuspended to make a 2X or 4X mix to accommodate a variety of sample input volumes.
LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG – lyophilized to be stored & shipped at room temperature
Same product performance as liquid format (#M3019)
Developed in collaboration with Fluorogenics Limited, a wholly owned subsidiary of New England Biolabs, Inc
Supplied as a lyophilized cake, the LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG enables sensitive detection of target RNA sequences in a room temperature-stable format. Rehydration is rapid, and the lyophilized cake will typically dissolve within 1-2 seconds after addition of water. Note that air from the cake will naturally degas from solution over ~20 seconds or after gentle vortexing.
RT-qPCR targeting human ß-actin was performed using either the LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) or Luna Probe One-Step 4X Mix with UDG (NEB #M3019) over an 8-log range of input template concentrations (1 μg – 0.1 pg Jurkat total RNA) with 4 replicates at each concentration, run on an ABI QuantStudio 6 Flex real-time instrument. Reactions (20 μl) included primers at 400 nM each and and probe at 200 nM, and followed the product recommended cycling conditions.
Lyophilized and liquid Luna RT-qPCR mixes demonstrate equivalent strong performance
LYOPHILIZED PRODUCTS FOR MDx OR OEM?
The ability to develop complex, yet simple to use lyophilized products enables us to provide a more complete solution for our customers, particularly those in the molecular diagnostics space. We have now extensive experience freeze-drying some of NEB’s most popular amplification products, effectively reducing the research and development timelines of custom products.
Aptamer-based enzyme control for room temperature setup and stability up to 24 hours
Highest fidelity multiplexing capacity supports use in ARTIC workflows, DNA arrays, cloning & sequencing etc.
Detect as low as 0.01 pg of human total RNA
Closed-tube system with cDNA synthesis and endpoint PCR amplification in a single protocol
LunaScript Multiplex One-Step RT-PCR Kit
E1555S/L
50/250 rxns
The LunaScript Multiplex One-Step RT-PCR Kit offers a streamlined protocol for cDNA synthesis and extremely high fidelity PCR amplification in a single reaction. The 5X reaction mix contains dNTPs and is optimized for robust multiple target detection in a simple workflow. The 25X enzyme mix features Luna WarmStart Reverse Transcriptase and Q5 Hot Start High-Fidelity DNA Polymerase offering the highest fidelity amplification available making it an ideal choice for next-generation sequencing, library construction DNA arrays, fragment analysis, electrophoresis and traditional cloning/sequencing workflows.
LunaScript Multiplex One-Step RT-PCR Kit – for virtually "error-free" multiplexing PCR
The LunaScript Multiplex One-Step RT-PCR Kit requires only a RNA template and gene-specific primers to enable multiplex cDNA target synthesis and amplification in a single reaction. Amplified cDNA products can be detected or identified by downstream applications including next-generation sequencing, DNA arrays, fragment analysis, electrophoresis and traditional cloning/sequencing workflows.
Optimized performance for real-time fluorescence and endpoint visualization detection methods
Set up reactions at room temperature with our unique dual WarmStart formulation
Reduce the risk of carryover contamination with thermolabile UDG and dUTP included in the mix
WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix with UDG
M1708S/L
100 / 500 rxns
The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is fully buffered and compatible with different sample types, enabling multiple detection methods including turbidity detection, real-time fluorescence detection, and end-point visualization such as colorimetric detection via a metal indicator (e.g., hydroxynaphthol blue). For real-time fluorescence detection, the master mix is available as a kit that includes 50X LAMP Fluorescent Dye. The inclusion of dUTP and thermolabile UDG enables carryover contamination prevention.
WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG)
The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with multiple detection methods.
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The core of your NGS Library Prep
NEW: Now also available as PCR-free workflow to eliminate the risk of PCR bias
Benefit from low input amount requirements, fewer PCR cycyles and extremely uniform GC-coverage in all applications
Save time with streamlined, modular workflows, reduced hands-on time, and automation compatibility
Learn one central workflow and apply it to a whole suite of different applications
As sequencing technologies continue to improve and applications expand, the need for compatibility with ever-increasing input amounts and sub-optimal sample quality grows. Reliability and high performance are critical, along with faster turnaround, higher throughput, and automation compatibility. NEBNext’s line of Ultra II DNA library prep reagents has expanded to include PCR-free workflows, powering high performance without the need for amplification. By eliminating the risk of PCR bias, libraries are a clearer reflection of biology. The NEBNext Ultra II workflow lies at the heart of NEB’s portfolio for next generation sequencing library preparation, with kits and modules for optimal flexibility. You can be assured your DNA libraries will be of the highest quality and yield, even when starting from extremely low input amounts.
NEBNext Ultra II reagents & kits: One central workflow for a wide range of applications – now featuring PCR-free workflows
The ULTRA II DNA WORKFLOW is available in convenient kit formats or modules:
TOOLS & RESOURCES
View performance data in our Technical Notes, which can be downloaded at NEBNextUltraII.com
View the NEBNext Ultra II DNA protocol video for protocol steps, and tips for optimization
Find hundreds of peer reviewed publications citing use of NEBNext Ultra II DNA on the product pages at NEB.com
"
– Dr. Jürgen Zimmermann, Senior Engineer - Automation, GeneCore EMBL Heidelberg, Germany
NEB is currently the only company, which is offering really "automation friendly" library preparation kits. The volumes of components are calculated to cover for unavoidable deadvolumes, reactant volumes are in range of most automated platforms. The stability of the NEBNext chemistry allows a broad range of automation strategies.
Use the Selection Chart below to determine which NEBNext Ultra II products best suit your needs:
Note: NEBNext Multiplex Oligos are available separately. Please visit www.neb.com/oligos for options.
NEBNext Ultra II DNA Library Prep Kit for Illumina
E7645S/L
24/96 rxns
NEBNext Ultra II DNA Library Prep with Sample Purification Beads
E7103S/L
NEBNext Ultra II FS DNA Library Prep Kit for Illumina
E7805S/L
NEBNext Ultra II FS DNA PCR-free Library Prep with Sample Purification Beads
E7435S/L
NEBNext Ultra II End Repair/dA-Tailing Module
E7546S/L
NEBNext Ultra II Ligation Module
E7595S/L
NEW: NEBNext Ultra II DNA PCR-free Library Prep Kit for Illumina
E7410S/L
NEW: NEBNext Ultra II DNA PCR-free Library Prep with Sample Purification Beads
E7415S/L
NEW: NEBNext Ultra II FS DNA PCR-free Library Prep Kit for Illumina
E7430S/L
NEW: NEBNext Ultra II FS DNA PCR-free Library Prep with Sample Purification Beads
NEBNext Ultra II FS DNA Module
E7810S/L
NEBNext FFPE DNA Repair Mix
M6630S/L
NEBNext Enzymatic Methyl-seq Kit
E7120S/L
NEBNext Enzymatic Methyl-seq Conversion Module
E7125S/L
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing
E7180S
24 rxns
Limited Offer: Ask your local distributor for a free sample of your favorite NEBNext kit!
Enzymatic MethylSeq (bisulfite-free)
Directional & non-directional RNA-seq
Enzymatic DNA Fragmentation System
Single Cell/ Low Input RNA Library Prep
SARS-CoV-2/ARTIC Surveillance Sequencing
Ready-to-use
We thought so! We’re confident that you will love our Quick-Load DNA ladders, with their sharp bands, ready-to-use format, and competitive pricing. In addition, our Quick-Load Purple ladders cast no UV shadow, so you’ll never miss a band.
Do you use DNA ladders in your daily lab work?
UV shadow comparison The innovative Gel Loading Dye, Purple (6X) (#B7024S) (Lane 1) included in the Quick-Load Purple Ladders does not cast a UV shadow over the underlying bands, unlike conventional Bromophenol-Blue containing gel loading dyes (Lane 2).
Free Poster For an overview of all available DNA, RNA and Protein standards, please request your free "Markers & Ladders" Poster from your local distributor.
Quick-Load Purple 1 kb Plus DNA Ladder
N0550S/L
250/750 gel lanes
Quick-Load 1 kb Plus DNA Ladder
N0469S
250 gel lanes
Quick-Load Purple 1 kb DNA Ladder
N0552S/L
125/375 gel lanes
Quick-Load 1 kb DNA Ladder
N0468S/L
Quick-Load 100 bp DNA Ladder
N0467S/L
Quick-Load Purple 50 bp DNA Ladder
N0556S
Quick-Load 1 kb Extend DNA Ladder
N3239S
125 gel lanes
Quick-Load Purple 100 bp DNA Ladder
N0551S/L
*Get special prices on all Quick Load (Blue and Purple) DNA Ladders as well as on NEB competent cells for cloning (page Cloning with Confidence). Campaign ends June 30th, 2022. Please ask your local distributor for details.
