Shop All cDNA Synthesis, Library, & Cloning Kits

Phusion Site-Directed Mutagenesis Kit (Thermo Scientific™)

Thermo Scientific Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid DNA. With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with T4 DNA Ligase. The resulting plasmid can be then transformed into any competent E. coli cells.

The optimal annealing temperature for Phusion DNA Polymerases may differ significantly from that of Taq-based polymerases.

For optimal results start by accurately calculating your Tm with our Tm calculator.


Highlights


• Robust and reliable exponential amplification method
• No requirements, such as special vectors, restriction sites, or methylation status for the target plasmid
• No need to destroy the starting template in a separate step
Phusion Hot Start II High Fidelity DNA Polymerase minimizes unwanted secondary mutations
• Amplification of large plasmids up to 10 kb
• Hot start modification of the polymerase prevents amplification of non-specific products and unwanted degradation of primers prior to first cycle of PCR
• T4 DNA Ligase included in the kit; no purification steps before or after ligation
• Compatible with all strains of competent E. coli cells

CloneMiner™ II cDNA Library Construction Kit (Invitrogen™)

The CloneMiner™ II cDNA Library Construction Kit is a second generation CloneMiner™ kit that enables rapid construction of highly representative cDNA libraries without restriction enzyme cloning. This innovative library construction technology combines SuperScript® III Reverse Transcriptase with Gateway® cloning technology, resulting in the discovery of previously unobtainable, full-length clones. The kit also avoids the use of time-consuming, inefficient ligation reactions, making library construction faster and offering better representation.

The CloneMiner™ II cDNA Library Construction Kit ensures:
• High primary titers
• Large average insert sizes
• The highest percentage of full-length genes
• Highly efficient cloning of cDNA to multiple destination vectors without the need of restriction enzyme digestion and ligation

How the CloneMiner™ II Kit Works:

• High yields of full-length cDNA: The CloneMiner™ II kit contains SuperScript® III for generating high cDNA yields. SuperScript® III Reverse Transcriptase (RT), a proprietary mutant of SuperScript® II RT, is active at 50°C and has a half-life of 220 minutes. It also has a point mutation that reduces RNase H activity, thereby decreasing RNA degradation during first-strand synthesis and increasing the percentage of full-length genes.

• Gateway® cloning technology avoids restriction enzyme cloning: Library construction is mediated by Gateway® Technology, a site-specific recombination system that eliminates the use of restriction enzymes and ligase in cloning. Each cDNA insert is flanked by specific att recombination sites (added during the cDNA synthesis steps) that recombine with complementary att sites present in Gateway® donor vectors to create entry clones. Entry clones are subsequently recombined with Gateway® expression vectors to create expression clones, effectively replacing the use of restriction enzymes and ligase. The resulting clones maintain the original orientation and reading frame enabling functional analysis of full-length genes and whole libraries.

How is This Kit Different From the Original CloneMiner™ Kit?
• The new kit incorporates a simplified protocol.
• To ensure high cDNA yields, SuperScript® II Reverse Transcriptase (RT) has been replaced by SuperScript® III Reverse Transcriptase.
• To enable reaction setup with fewer pipetting steps, Gateway® BP Clonase® Enzyme Mix has been replaced by Gateway® BP Clonase® II Enzyme Mix, which contains enzymes and buffer in a single mix.

FirstChoice™ RLM-RACE Kit (Invitrogen™)

The FirstChoice® RLM-RACE Kit is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA—no rRNA, tRNA or degraded RNA—and facilitates the cloning of sequences from the 5' ends of messages.

Features of the FirstChoice® RLM-RACE Kit:

• From RNA to PCR product in less than a day
• Yields single, specific product from rare transcripts
• Selects 5' and/or 3' ends of true messages
• Efficient—all enzymatic reactions are optimized to ensure detection of even the rarest mRNA

RACE
Rapid amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of sequence from the 5'-ends of messages. The FirstChoice® RLM-RACE Kit is a major improvement to the basic RACE protocol.

Selects full-length mRNA—no rRNA, tRNA or degraded mRNA
In the FirstChoice® RLM-RACE procedure, full-length mRNAs are selected by treating total or poly(A) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules which contain free 5'-phosphates (ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA). Full-length mRNAs are unaffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the full-length mRNA leaving a 5'-monophosphate. A synthetic RNA adapter is ligated to the RNA population—only molecules containing a 5'-phosphate, the uncapped, full-length mRNAs, will accept the adapter. Random-primed, reverse transcription reactions and nested PCR are then performed to amplify the 5'-end of your specific transcript.

From RNA to PCR in less than a day
Each step of the FirstChoice® RLM-RACE procedure has been optimized, so you can complete all the enzymatic reactions and even start PCR in no more than 5 hours. The FirstChoice® RLM-RACE Kit contains reagents and enzymes to produce 5 RLM-RACE-ready cDNA preparations, in addition to primers and nested RACE adapter primers for 100 PCR reactions. Reverse transcription reagents are also included. Each kit contains control RNA and primers to test the kit's performance along with a detailed Instruction Manual. SuperTaq™ Thermostable Taq DNA Polymerase is available separately. For optimal amplification of fragments ≥1 kb, use SuperTaq-Plus. Also included are a 3' RACE Adapter and Primers for 3' RACE.

High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems™)

The High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single-stranded cDNA in a single 20 µL reaction. This particular kit also includes 1 tubes of RNase inhibitor (200 µL).

The High Capacity cDNA Reverse Transcription Kit delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg total RNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction. Downstream applications include real-time PCR, standard PCR, and microarrays. The kit is ideal for generating cDNA archives. Features of this kit include:

• Linear target amplification for real-time PCR
• Higher yields and precision than other cDNA synthesis kits—at a fraction of the cost
• 10-fold greater dynamic range than other kits

Extensively tested with a variety of templates
Quantitative first-strand synthesis of all RNA species is achieved with the use of the random primers. The kit has been extensively tested with a variety of RNA templates, including GC- and AU-rich targets and RNAs expressed at low levels. cRNA can also be efficiently generated from in vitro transcription of cDNA.

GeneArt™ High-Order Genetic Assembly Systems, with yeast growth media (Invitrogen™)

The GeneArt® High-Order Genetic Assembly System is a highly efficient kit for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 Kbp in length, into any vector. The system relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This greatly reduces the in vitro handling of DNA and eliminates the need for enzymatic treatments, such as restriction and ligation, while allowing for precise fusions of DNA sequences. The kit contains materials for the transformation and purification from yeast, including yeast selective media, and competent E. coli for plasmid amplification of correct clones.

Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt® Seamless Cloning and Assembly Kit (cat # A13288).

Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt® High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.

Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit.

Considerations for Choosing a Cloning Vector
The GeneArt® High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt® pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt® High-Order Vector Conversion Cassette (cat#A13291).

Cloning Efficiency
In GeneArt® High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ are the following:
• >90% for 5 DNA fragments of 10 Kb each
• >90% for 10 DNA fragments of 5 Kb each
• >50% for 10 DNA fragments of 10 Kb each

Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
• >90% for 1 fragment of 10 Kb
• >75% for 2 DNA fragments of 10 Kb each

Design Your Cloning in silico
A key step in GeneArt® High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.

Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase (Thermo Scientific™)

Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase is a convenient system optimized for cDNA synthesis in 2-step quantitative RT-PCR (RT-qPCR) applications. It provides a dramatically simplified workflow that combines genomic DNA elimination and cDNA synthesis into one-tube procedure. The kit contains a novel double-strand specific DNase (dsDNase) engineered to remove contaminating genomic DNA from RNA preps in 2 minutes without damage to quality or quantity of RNA. Highly specific activity towards double-stranded DNA ensures that single-stranded DNA (such as cDNA and primers) is not cleaved and dsDNase treated RNA can be directly added to reverse transcription.

For the first strand cDNA synthesis the kit uses Maxima Reverse Transcriptase (RT), an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features high thermostability, robustness and increased cDNA synthesis rate compared to wild type M-MuLV RT. The Maxima First Strand cDNA Synthesis Kit for RT-qPCR is capable of reproducible cDNA synthesis from a wide range of total RNA amounts (1 pg to 5 µg) at elevated temperatures (42 to 65°C). The synthesis reaction can be completed in 15 to 30 minutes. Components of the Maxima First Strand cDNA Synthesis Kit for RT-qPCR are pre-mixed to save time and to reduce the possibility of pipetting errors.

Highlights

• Integrated gDNA removal step
• High yields of full length cDNA up to 20 kb.
• Efficient cDNA synthesis at wide temperature range from 42°C to 65°C.
• Increased synthesis rate—complete cDNA synthesis in 15-30 minutes.
• High sensitivity and specificity.

Applications

• 2-step RT-PCR
• 2-step RT-qPCR

Maxima First Strand cDNA Synthesis Kit with dsDNase contains
• Maxima Enzyme Mix
• 5X Reaction Mix
• dsDNase
• 10X dsDNase Buffer
• nuclease-free water

Additional Information about Reaction Components
The Maxima Enzyme Mix contains Maxima Reverse Transcriptase and Thermo Scientific RiboLock RNase Inhibitor,. The recombinant RiboLock RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B and C at temperatures up to 55°C.

5X Reaction Mix contains the remaining reaction components: reaction buffer, dNTPs, oligo(dT)18 and random hexamer primers.

Water, nuclease-free is provided for reaction set-up and dilution of sample RNA. The absence of endo-, exodeoxyribonucleases, ribonucleases and phosphatases has been confirmed by appropriate quality tests.

Related Products
Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase
Maxima First Strand cDNA Synthesis Kit for RT-qPCR
Maxima First Strand cDNA Synthesis Kit Reaction Mix (5X)

SuperScript™ IV First-Strand Synthesis System (Invitrogen™)

The SuperScript® IV First-Strand Synthesis System for RT-PCR is optimized for synthesis of first-strand cDNA from purified poly(A)+ or total RNA. The kit includes SuperScript® IV Reverse Transcriptase (RT), a proprietary MMLV mutant that produces robust and reliable cDNA synthesis in RT reactions. The SuperScript® IV synthesis system is significantly improved over the SuperScript® III-based system in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous system, including increased thermostability, highly efficient full-length cDNA synthesis, and reduced RNase activity. The SuperScript® IV system is designed to provide reliable, consistent, and fast cDNA synthesis in the presence of inhibitors found in a wide variety of samples that cause other currently available RTs to perform inefficiently.

The SuperScript® IV First-Strand Synthesis System contains all components needed for RT reactions, plus an additional control gene and primers, and provides the flexibility to customize the RT set-up to fit the needs of your application. The SuperScript® IV synthesis system is the top choice for performance and flexibility for RT-PCR applications. We recommend it for all RT-PCR applications, especially when reproducibility and reliability is the primary concern and when inhibitors in the RNA sample may interfere with cDNA synthesis, leading to biases in gene expression studies.

Features of the SuperScript® IV First-Strand Synthesis System include:
• Significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis
• Robust and specific cDNA synthesis in a wide range of sample types
• A faster reverse transcriptase reaction that reduces incubation time from >50 min to 10 min
• Significantly better processivity compared to SuperScript® III RT

Enzyme source
Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability and half-life, processivity, inhibitor resistance and to reduce RNase H activity.

Performance and quality testing
Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product.

Unit definition
One unit of SuperScript® IV RT is the amount of enzyme required to incorporate 1 nmole of deoxyribonucleotide into acid-precipitable material in 10 min at 37°C using poly(A) oligo(dT)12-18 as a template/primer.

Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [3H]-dTTP, 0.4 mM poly(A) oligo (dT)12-18 and enzyme in 20 μL for 10 min at 37°C.

High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™)

The High Capacity RNA-to-cDNA Kit is a streamlined reverse transcription kit designed for optimum performance with TaqMan Gene Expression Master Mix, Power SYBR Green PCR Master Mix, and other PCR enzymes.

Features of this kit include:
Fast—short reaction time (typically 30–60 min)
Convenient—easy workflow with few pipetting steps (2 tubes)
Reliable—reverse transcription of both abundant and limited targets
Flexible—optimized 2-step protocol, enabling multiple PCR reactions from a single reverse transcription reaction

Streamline your cDNA synthesis while maintaining detection sensitivity
The components of the two-tube kit work together to provide sensitive and specific reverse transcription across a broad range of template amounts. Amplification of a dilution series of the RNA concentration reference standard demonstrates exceptional reverse transcription and PCR efficiency across 11 orders of magnitude. This wide dynamic range allows one set of reaction conditions to detect transcripts from highly active as well as weakly expressed genes. The two-tube formulation reduces reaction time and requires fewer pipetting steps than with the High Capacity cDNA Reverse Transcription Kit, while maintaining high performance.

GeneArt™ Type IIs Assembly Kit, Aar I (Invitrogen™)

The GeneArt® Type IIs Assembly Kit, Aar I, provides seamless cloning and assembly of up to 8 DNA fragments by simultaneous cleavage and ligation in a single reaction. The kit uses a technology similar to Golden Gate cloning with the type IIs restriction enzyme Aar I and can be used to assemble multiple fragments in a pre-determined order into any compatible vector. Since type IIs assembly is not based upon homologous recombination, there is minimal risk of rearrangements and minimal sequence confirmation of your final construct is required.

The kit is offered in three versions, each with a different type IIs restriction enzyme (Aar I, Bsa I, or Bbs I). All versions include all-in-one enzyme mix, cloning vector, and cloning controls. Our GeneArt® Primer and Construct Design Tool should be used to determine the appropriate GeneArt® assembly kit for your fragments; it provides an easy-to-use interface to design your construct and create and order primers, should they be required.

• Assemble multiple DNA fragments in any order, into any compatible vector, without scars
• Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
• Use for assembly of TALs, gene variants, and repetitive sequences
• Create your own cloning and expression vectors with custom vector elements
• Minimize sequence confirmation of final construct
• Pick from three type IIs enzymes

Type IIs Cloning
GeneArt® Type IIs Cloning is a simple, two-step process, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. The type IIS restriction endonuclease Aar I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Aar I recognition site. The ends of your DNA fragment can be designed to be flanked by the Aar I restriction site such that digestion of the fragments removes the recognition site and generates complementary overhangs that can be ligated seamlessly, creating a junction that lacks the original site. The kit includes a destination vector, called pType IIs, which is optimized for this approach.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Type IIs Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA elements, the total size of the final molecule (? 10 kb), and the quality and specificity of the fragment.

Typical cloning efficiencies for different numbers of fragments cloned into pType IIs are:

• >95% for 5 fragments of 1 kb each
• >60% for 8 fragments of 1 kb each
• >85% for 2 identical fragments of 1 kb each

in silico Cloning Design Support
A key step in GeneArt® Type IIs Cloning is the correct design of fragments and oligos with the appropriate recognition sites and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide the GeneArt® Primer and Construct Design Tool to help you design your experiment in silico. The tool recommends the correct GeneArt® kit for your assembly, checks for compatibility of the experimental design with the product specifications, designs DNA oligos, if needed, and presents the user with a graphical representation of the vector, as well as a downloadable annotated sequence in GenBank format that is compatible with VectorNTI® software.

Additional recommended products:

• Zero Blunt® TOPO® PCR Cloning Kit, without competent cells (Cat. No. 450245)
• Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Cat. No. K280020)
• One Shot® MAX Efficiency™ DH10B™ T1 Phage Resistant Cells (Cat. No. 12331-013)

Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific™)

Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR is a convenient system optimized for cDNA synthesis in 2-step quantitative RT-PCR (RT-qPCR) applications. The kit uses Maxima Reverse Transcriptase (RT), an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features high thermostability, robustness and increased cDNA synthesis rate compared to wild type M-MuLV RT.

The Maxima First Strand cDNA Synthesis Kit for RT-qPCR is capable of reproducible cDNA synthesis from a wide range of total RNA amounts (1 pg to 5 µg) at elevated temperatures (42 to 65°C).The synthesis reaction can be completed in 15 to 30 minutes. Components of the Maxima First Strand cDNA Synthesis Kit for RT-qPCR are pre-mixed to save time and to reduce the possibility of pipetting errors.

Highlights

• High yields of full length cDNA up to 20 kb
• Efficient cDNA synthesis at wide temperature range from 42°C to 65°C
• Increased synthesis rate—complete cDNA synthesis in 15-30 minutes
• High sensitivity and specificity

Applications

• 2-step RT-PCR
• 2-step RT-qPCR

Maxima First Strand cDNA Synthesis Kit for RT-qPCR contains
• Maxima Enzyme Mix
• 5X Reaction Mix
• nuclease-free water

Additional Information about Reaction Components
The Maxima Enzyme Mix contains Maxima Reverse Transcriptase and Thermo Scientific RiboLock RNase Inhibitor,. The recombinant RiboLock RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B and C at temperatures up to 55°C.

5X Reaction Mix contains the remaining reaction components: reaction buffer, dNTPs, oligo(dT)18 and random hexamer primers.

Water, nuclease-free is provided for reaction set-up and dilution of sample RNA. The absence of endo-, exodeoxyribonucleases, ribonucleases and phosphatases has been confirmed by appropriate quality tests.

Related Products
Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase
Maxima First Strand cDNA Synthesis Kit for RT-qPCR
Maxima First Strand cDNA Synthesis Kit Reaction Mix (5X)

TaqMan™ Reverse Transcription Reagents (Invitrogen™)

TaqMan® Reverse Transcription Reagents provide the necessary reagents for reverse transcriptase-PCR (RT-PCR). Random hexamers, oligo (dT)16, and reverse primers are included for cDNA synthesis flexibility. Advantages of TaqMan® Reverse Transcription Reagents:

• Contain MultiScribe® Reverse Transcriptase, a recombinant moloney murine leukemia virus reverse transcriptase (MMLV RT)
• Random hexamers and oligo (dT)16 included for flexibility
• Individual components provide versatility in assay set-up
• Compatible with two-step RT-PCR reactions

Using TaqMan® Reverse Transcription Reagents
TaqMan® Reverse Transcription Reagents provide all the necessary components to perform the reverse transcription of RNA to cDNA. For two-step RT-PCR, random hexamers, oligo (dT)16, and sequence-specific reverse primers can all be used. The choice of primers for reverse transcription is best made after experimentally evaluating all three priming systems.

Maxima First Strand cDNA Synthesis Kit Reaction Mix (5X) (Thermo Scientific™)

Thermo Scientific Maxima 5X Reaction Mix is a component of the Maxima First Strand cDNA Synthesis Kits for RT-qPCR (K1671/K1672/K1641/K1642) and may be purchased separately. The 5X Reaction Mix is comprised of reaction buffer, dNTPs, oligo (dT)18 and random hexamer primers.

Related Products
Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase
Maxima First Strand cDNA Synthesis Kit for RT-qPCR

GeneArt™ Site-Directed Mutagenesis System (Invitrogen™)

The GeneArt® Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to generate mutations on a target construct in vitro in less than three hours. This system replaces the popular GeneTailor™ Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed mutagenesis products currently available on the market. This product brings our mutagenesis technology to the next level. Note: A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.

Powerful – Make substitutions, deletions, or insertions of up to 12 nucleotides in plasmids up to 14 Kb
Efficient – Enables you to obtain your desired mutant the first time; over 90% correct mutants for a 3 Kb plasmid
Fast – Have your mutated plasmid DNA in less then 3 hours with the simple, minimal handling protocol
Versatile – Use plasmids of many sizes, and DNA isolated from any source, with no need for specialized vectors, host strains, or restriction sites

Simple Creation of Desired Mutants
Creating mutants with the GeneArt® Site-Directed Mutagenesis System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli. Simply incorporate the mutation or mutations (up to 12 nucleotides), that you want into primers, and after PCR, recombination, and transformation you get vectors with only the mutations you desired with up to 90% efficiency. The template vector that you add mutations to can be isolated from any source and up to 14Kb in size. For creating complete constructs, or for joining large pieces of unrelated DNA see our GeneArt® Seamless Cloning and Assembly Kit (cat# A13288) or our GeneArt® High-Order Genetic Assembly System (cat#A13286).

Optimized Mutagenesis Protocol
The GeneArt® Site-Directed Mutagenesis System has been optimized for efficiency and simplicity. The DNA methylation and amplification steps are combined into a single reaction with no need for an in vitro DpnI digestion step. After methylation and amplification, a 10 minute in vitro recombination reaction of the amplified PCR products increases the colony output by 3 to 10 fold; resulting in a higher mutagenesis efficiency. A final transformation of the mutated DNA into DH5α™ E.coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. No purification steps or additional digestions are needed. Individual colonies can be selected the following day to verify mutations.

Cells-to-cDNA™ II Kit (Invitrogen™)

The Ambion® Cells-to-cDNA™ II Kit (patent pending) produces cDNA from cultured mammalian cells in less than 2 hr. No RNA isolation is required. This kit contains sufficient reagents for 40 reactions and is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation.

• No RNA purification required
• From cells in culture to cDNA in less than 2 hr
• Detect rare messages in as few as 1 cell
• Ideal for labs not equipped for RNA isolation

Ideal for RT-PCR Applications
The Cells-to-cDNA™ II Kit (patent pending) integrates RNase inactivation and DNase I treatment into an RT-PCR compatible cell lysis buffer, eliminating RNA isolation altogether. Most cell lysis buffers contain strong denaturants that if carried over into enzymatic reactions would inhibit or inactivate most enzymes. If a lysis buffer without strong denaturants is used, endogenous RNase can quickly degrade cellular RNA. The Cells-to-cDNA™ II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA™ II is compatible with both one-step and two-step RT-PCR protocols.

Quantitative—Linear Results and No Detectable DNA Contamination
To illustrate the quantitative, linear response of Cells-to-cDNA™ II to variations in input cell number, a real-time quantitative RT-PCR experiment was performed using the ABI 7700 Sequence Detection System. Plotting cycle threshold (Ct) versus cell concentration for GAPDH yielded a standard curve with a correlation of 0.99. Thus, Cells-to-cDNA™ II yields linear results for 1 to 10,000 cells/ µLin a real-time RT-PCR two-step assay. It should also be noted that the minus-template PCR control for the GAPDH experiment was negative, indicating complete removal of genomic DNA.

Simple Procedure
The kit comes with everything you need to go from cultured cells to PCR-ready cDNA in less than 2 hr; no RNA isolation is required. Cells are first washed with PBS and then resuspended in the Cell Lysis Buffer. A brief heating simultaneously lyses the cells and inactivates RNases. An optional DNase digestion is then performed to degrade genomic DNA, followed by a heat inactivation step. A fraction of the cell lysate is then used in a reverse transcription reaction with the included reagents.

Accessory Products:
SuperTaq™ Thermostable Taq DNA Polymerase is recommended and available separately (SKU#s AM2050 and AM2052). For optimal amplification of fragments greater than 1 kb, use SuperTaq™ Plus Thermostable Taq DNA Polymerase (SKU#s AM2054 and AM2056).

Arcturus™ RiboAmp™ HS PLUS cDNA Kit (Applied Biosystems™)

The ARCTURUS® RiboAmp® HS PLUS cDNA Kit permits quantitative Real-Time PCR (qRT-PCR) from as little as 100pg of total RNA. The kit uses random primers to enable robust reverse transcription across a broader transcript length.

Integrated Systems for Microgenomics
The ARCTURUS® Complete System for Microgenomics® offers an integrated solution for preparing small quantities of RNA for gene expression analysis. The System features the ARCTURUSXT™ Laser Capture Microdissection (LCM) Instrument to procure pure cell populations, the PicoPure® RNA Isolation Kit to extract and purify RNA, and the RiboAmp® PLUS Kit for reverse transcription and linear amplification of RNA. ARCTURUS® Systems for Microgenomics enable accurate and sensitive qRT-PCR and microarray assays that reveal molecular otherwise obscured in whole tissue samples or cell mixtures.

For Research Use Only. Not for use in diagnostics procedures.