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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.

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

Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific™)

Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit is a complete system for highly efficient synthesis of first strand cDNA. The kit uses the Maxima H Minus Reverse Transcriptase (RT), which is an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features the highest thermostability among the derivatives of M-MuLV RT and lacks RNase H activity. The Maxima H Minus First Strand cDNA Synthesis Kit allows synthesis of long cDNAs up to 20 kb at elevated temperatures (up to 65°C), superseding other systems' abilities to produce full-length cDNA. Due to increased synthesis rates the reaction can be completed in 30 minutes.

Highlights

• Increased reaction temperatures—the first strand of cDNA can be synthesized within the 42 to 65°C temperature range
• High yields of full-length first strand cDNA—with RNA templates up to 20 kb
• Flexible priming—oligo(dT)18, random hexamer, or gene-specific primers

Applications

• First Strand cDNA synthesis for RT-PCR
• Construction of cDNA libraries
• Generation of probes for hybridization
• Antisense RNA synthesis

Includes

• Maxima H Minus First Strand cDNA Synthesis Kit contains Maxima H Minus Enzyme Mix, Oligo(dT)18 and Random hexamer primers, 5X RT Buffer, dNTP Mix, and nuclease-free water.

Additional information about reaction components

• Maxima H Minus Enzyme Mix contains Maxima H Minus Reverse Transcriptase and RiboLock RNase Inhibitor. RiboLock RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B and C at temperatures up to 55°C.
oligo(dT)18 and random hexamer primers are supplied with the kit. Random hexamer primers bind non-specifically and are used to synthesize cDNA from all RNAs in a total RNA population. The oligo(dT)18 primer selectively anneals to the 3'-end of poly(A) RNA, synthesizing cDNA only from poly(A) tailed mRNA. Gene-specific primers may also be used with the kit to prime synthesis from a specified sequence.
10 mM dNTP Mix is a premixed aqueous solution of dATP, dTTP, dCTP, and dGTP.
Nuclease-free water is provided for reaction set-up and dilution of sample DNA. The absence of endo-, exodeoxyribonucleases, ribonucleases, and phosphatases has been confirmed by appropriate quality tests.

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Maxima H Minus First Strand cDNA Synthesis Kit, with dsDNase
Maxima H Minus First Strand cDNA Synthesis Kit

RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific™)

Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit uses RevertAid Reverse Transcriptase (RT), a recombinant M-MuLV RT which maintains activity at 42-50°C and is suitable for synthesis of cDNA up to 13 kb. RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA templates from degradation.
The RevertAid First Strand cDNA Synthesis Kit is supplied with both oligo(dT)18 and random hexamer primers. The oligo(dT)18 primer anneals selectively to the poly(A) tail of mRNA. Random hexamer primers do not require the presence of the poly(A) tail, therefore, they can be used for transcription of the 5'-end regions of mRNA or cDNA synthesis of RNA species lacking a poly(A) tail (e.g., microRNAs). Gene-specific primers may also be used with the kit.

Highlights

• Full-length first strand cDNA up to 13 kb
• Optimum reaction temperature 42°C
• Complete kit—all the components for the RT reaction are included

Applications

• First strand cDNA synthesis for RT-PCR and RT-qPCR
• Construction of full length cDNA libraries
• Antisense RNA synthesis

Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells (Thermo Scientific™)

This package includes a Phusion Site-Directed Mutagenesis Kit and DH10B Competent Cells that are optimized for use with the kit.

Phusion Site-Directed Mutagenesis Kit
The Thermo Scientific Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions into 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 the E.coli cells. DH10B Competent Cells are recommended for use with the kit.

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.

• 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

DH10B Competent Cells
Thermo Scientific DH10B Competent Cells are high efficiency, chemically competent E.coli cells. The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently. These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors. Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG.

• High transformation efficiency: >1x109 cfu/µg pUC19 DNA
• Suitable for routine and high-throughput cloning and mutagenesis applications
• Convenient two reactions per vial packaging
• S.O.C. Outgrowth medium included

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Thermo Scientific Phusion Site-Directed Mutagenesis Kit without competent cells

Verso cDNA Synthesis Kit (Thermo Scientific™)

Thermo Scientific Verso cDNA synthesis Kit provides robust transcription of RNA to create a complete cDNA pool. The Verso system achieves robust and sensitive reverse transcription through the combination of a high affinity RT enzyme, a unique RNA priming method, and an optimized buffering system. The Verso RT enzyme has high RNA template affinity and reduced RNase H activity, to transcribe even sections with high secondary structure. The included anchored oligo dT priming method further enhances sensitivity by increasing transcription efficiency, and the cDNA synthesis buffer has been optimized to achieve a full and diverse cDNA pool.

Highlights

• Highly sensitive with a broad dynamic range: can reverse transcribe RNA from an input range of 1pg to 1µg
• Efficient RT reaction allows for 75% shorter protocol times (down to 30 minutes)
• Unique priming strategy for increased efficiency and broad template flexibility
• High affinity RT enzyme effectively transcribes through difficult RNA sequences
• Wide working temperature range (42°C to 57°C) for success with GC-rich and other difficult templates
• Included RT Enhancer prevents genomic DNA carryover, eliminating the need for separate DNase I treatment
• RNase Inhibitor included
• Can be paired with any PCR Kit for complete 2-Step RT-(q)PCR

Applications

• 2-step RT-(q)PCR
• Single stranded cDNA synthesis

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GeneRacer™ Kit with AMV RT and TOPO TA Cloning™ Kit for Sequencing (Invitrogen™)

GeneRacer® is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5´ and 3´ cDNA ends. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts up to 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequence

The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.

How GeneRacer® Works

The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (1,2). Figure 1 outlines how the GeneRacer® Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.

Sensitivity and Length

To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5fi cDNA end, the 5fi ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified (Figure 2).

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.

SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning with Gateway™ Technology (Invitrogen™)

The SuperScript® Plasmid System is designed for synthesis of double-stranded cDNA from a purified mRNA population. The cDNA is suitable for directional cloning into the pSPORT1 vector or the pCMV

• SPORT6 plasmid vector for subsequent generation of cDNA libraries and use of Gateway® Technology. The SuperScript® Plasmid System:
• Results in greater cDNA yields and increased full-length cDNAs with SuperScript® II RT
• Offers a simplified primer-adapter strategy for directional cloning
• Offers one-tube format for first- and second-strand reactions, improving cDNA yields
• Fractionates cDNA using efficient and convenient pre-packed columns
• Ligates inserts into a pre-cut Not I-Sal I vector prepared to minimize background

SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen™)

The SuperScript® III First-Strand Synthesis SuperMix is an optimized SuperMix formulation for first-strand cDNA synthesis from purified poly(A)+ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 0.1 pg to 5 µg of total RNA.

Using the SuperScript® III First-Strand Synthesis SuperMix
The kit includes SuperScript® III/RNaseOUT™ Enzyme Mix, 2X First-Strand Reaction Mix, and Annealing Buffer. SuperScript® III Reverse Transcriptase, included in the Enzyme Mix, is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 45–55°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript® III RT is not significantly inhibited by ribosomal and transfer RNA, it can be used to synthesize cDNA from total RNA. RNaseOUT™ Recombinant RNase Inhibitor is included in the Enzyme Mix to safeguard against degradation of target RNA due to ribonuclease contamination. The 2X First-Strand Reaction Mix includes 10 mM MgCl2 and 1 mM of each dNTP in a buffer formulation that has been optimized for first-strand synthesis of cDNA. The Annealing Buffer is used in the initial template-primer annealing step. Separate tubes of oligo(dT)20 and random hexamers are also provided. cDNA synthesis can be performed using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer.

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.

Maxima H Minus Double-Stranded cDNA Synthesis Kit (Thermo Scientific™)

Thermo Scientific Maxima H Minus Double-Stranded cDNA Synthesis Kit is a complete system for efficient synthesis of double-stranded cDNA from total RNA or mRNA. First- and second-strand cDNA synthesis reactions are performed in the same tube without the need for intermediate organic extraction or ethanol precipitation steps. This convenient one-tube format speeds up the synthesis procedure and maximizes cDNA recovery. The kit contains premixed components to reduce the number of pipetting steps necessary to complete the procedure.

Highlights

• Efficient synthesis of full length double-stranded cDNA
• Fast—procedure completed in less than 2 hours
• Convenient—premixed components
• Complete—includes all primers, controls and residual RNA removal reagents

Applications

• Full-length double-stranded blunt-end cDNA synthesis for cloning
• Double-stranded cDNA library construction

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).

SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen™)

SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR provides the high-temperature capability of SuperScript® III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first-strand cDNA for use in real-time quantitative RT-PCR (qRT-PCR). The simple, time-saving reaction set-up uses just two tubes: a 2X Reaction Mix and an Enzyme Mix.

Enzyme mix
SuperScript® III Reverse Transcriptase, included in the RT Enzyme Mix, is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 42–60°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript® III RT is not significantly inhibited by ribosomal and transfer RNA, it can be used to synthesize cDNA from total RNA. RNaseOUT™ Recombinant Ribonuclease Inhibitor, also included in the enzyme mix, is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation.

Reaction mix
The 2X RT Reaction Mix includes oligo(dT)20, random hexamers, MgCl2, and dNTPs in a buffer formulation that has been optimized for qRT-PCR. E. coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA:RNA hybrid molecule after first-strand synthesis. This has been shown to increase sensitivity in qRT-PCR.

Using SuperScript® III First-Strand Synthesis SuperMix
This SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT-PCR, with a broad dynamic range that supports accurate quantification of high-copy mRNA from up to 1 µg of total RNA. Reagents are provided for 50 or 250 RT reactions of 20 µL each.

SuperScript™ Choice System for cDNA Synthesis (Invitrogen™)

The SuperScript® Choice System is designed for synthesis of double-stranded cDNA from a purified mRNA population. Advantages of the SuperScript® Choice system include:

• The advanced capabilities of SuperScript® II RT resulting in greater cDNA yields and increased full-length cDNAs (1,3)
• Choice of primers for first-strand synthesis (2)
• One-tube format improving cDNA yield (4,5)
• cDNA is size-fractionated using an efficient and convenient pre-packed column
• One reaction converts up to 5 µg of mRNA into size-fractionated, EcoR I-adapted cDNA, ready for ligation into any EcoR I-digested vector

Performance and Quality Testing: Yields of the first- and secondstrand cDNA synthesis reactions and percentage of full-length double-stranded cDNA are assessed using the control RNA.

GeneRacer™ Kit with SuperScript™ III RT and TOPO TA Cloning™ Kit for Sequencing (Invitrogen™)

The GeneRacer™ Kit provides a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequence from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. The kit ensures the amplification of only full-length transcripts via elimination of truncated messages from the amplification process. RACE PCR products can be quickly and easily cloned using either the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (blunt-end PCR products) or the TOPO TA Cloning® for Sequencing Kit (PCR products with 3' A-overhangs). Using the protocols provided, the cDNA ends of rare (30 copies/cell) and long (9 kb) transcripts can be amplified and sequenced starting from 1 µg of total RNA. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts at least 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequenceSuperScript® III RT
The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.How the GeneRacer® Kit works
The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (see figure). The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps, the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.Sensitivity and length
To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5´ cDNA end, the 5´ ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell, were amplified (see figure).