Shop All cDNA Synthesis, Library, & Cloning Kits

GeneArt™ Type IIs Assembly Kit, Bsa I Invitrogen™

The GeneArt® Type IIs Assembly Kit, Bsa 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 Bsa 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 Bsa I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Bsa I recognition site. The ends of your DNA fragment can be designed to be flanked by the Bsa 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 (up to 13 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)

SuperScript™ Double-Stranded cDNA Synthesis Kit Invitrogen™

The SuperScript® Double-Stranded cDNA Synthesis Kit contains all of the reagents (except an oligo (dT)-containing primer) necessary to make high-quality, double-stranded cDNA from total RNA or poly A+-selected RNA (mRNA). The advanced capabilities of SuperScript® II Reverse Transcriptase, with its reduced RNase H activity, maximize the yield of full-length cDNA, as well as that of overall cDNA synthesis. Features of the SuperScript® Double-Stranded cDNA Synthesis Kit include:

Reliability—each buffer, reagent, and enzyme in the kit is of the same high quality you expect from us
Performance—SuperScript® II is an enzyme you can trust for high-yield, high-quality, general-purpose, first-strand synthesis.

Simple, reliable, and trouble-free
The SuperScript® Double-Stranded cDNA Synthesis Kit offers a simple, reliable, and trouble-free method by which RNA templates can be converted to cDNA for use in cloning and library construction experiments. The reactions described in the SuperScript® Double-Stranded cDNA Synthesis Kit protocol are designed to convert 25–50 µg total RNA, or 0.2–5 µg mRNA, into first- and second-strand cDNA (supply your own primer). Enjoy the benefits of a tested protocol and reagents of consistent quality. To get the best possible cDNA product using this kit, we recommend the use of a high-quality RNA substrate prepared using TRIzol® RNA Isolation Reagents.

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).

Maxima™ H Minus cDNA Synthesis Master Mix, with dsDNase Thermo Scientific™

Thermo Scientific Maxima H Minus cDNA Synthesis Master Mix with dsDNase provides a simple workflow that combines genomic DNA elimination and cDNA synthesis in a one-tube procedure. The cDNA reaction components are pre-mixed into a complete master mix that is convenient to use, reduces pipeting steps, and is optimized for cDNA synthesis in two-step quantitative RT-PCR (RT-qPCR) applications. Maxima H Minus cDNA Synthesis Master Mix is optimized for reproducible cDNA synthesis at elevated temperatures (50–65°C) within 30 minutes.

Features include:
• One-tube master mix for reduced pipetting steps and enhanced consistency in RT-qPCR results
• Increased RT efficiency across a wide range of input RNA amounts and gene targets
• High thermostability to allow RT reactions at 50 to 65°C temperature range
• Integrated step of genomic DNA removal from RNA samples

The master mix contains Maxima H Minus Reverse Transcriptase (RT) and RiboLock RNase Inhibitor. Maxima H Minus RT is an advanced enzyme derived from M-MuLV RT by in vitro evolution. The enzyme features high thermostability, robustness, processivity, increased cDNA synthesis rate, and lack of RNase H activity. The recombinant RiboLock RNase Inhibitor effectively protects RNA template from degradation by RNases A, B, and C at temperatures up to 55°C. The master mix also contains reaction buffer, dNTPs, oligo (dT)18, and random hexamer primers. A separate tube of No-RT control mix is provided for convenient RT negative control.

The included double-strand specific DNase (dsDNase) allows removal of genomic DNA from RNA samples in 2 minutes without affecting the quality or quantity of RNA. This dsDNase is also available separately (Cat. No. EN0771).

Additional information about reaction components
• The No RT Control mix contains all components in the Maxima H Minus cDNA Synthesis Master Mix except Maxima H Minus RT. The presence of a PCR product in the No RT Control reaction indicates that the reaction is contaminated with DNA. To further enhance genomic DNA elimination efficiency, template RNA incubation with the included dsDNase is strongly recommended.
• The dsDNase is used for rapid and safe removal of contaminating genomic DNA from RNA samples. dsDNase is easily inactivated by moderate heat treatment (55°C). It allows for a dramatically simplified workflow that combines genomic DNA elimination and cDNA synthesis into one-tube procedure.
• Nuclease-free water is provided for reaction set-up and dilution of sample DNA. The absence of exodeoxyribonucleases, ribonucleases, and phosphatases has been confirmed by appropriate quality tests.

Related products
Maxima H Minus First Strand cDNA Synthesis Kit, with dsDNase
Maxima H Minus First Strand cDNA Synthesis Kit
Maxima H Minus Reverse Transcriptase
dsDNase

GeneArt™ Type IIs Assembly Kit, Bbs I Invitrogen™

The GeneArt® Type IIs Assembly Kit, Bbs 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 Bbs 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 Bbs I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Bbs I recognition site. The ends of your DNA fragment can be designed to be flanked by the Bbs 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)

GeneArt™ Gibson Assembly HiFi Cloning Kit, chemically competent cells Invitrogen™

The GeneArt Gibson Assembly HiFi Cloning Kit enables DNA assembly and cloning via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 15–60 minute isothermal reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility of this approach is suitable for small and large DNA constructs and includes both single and multiple inserts. The resulting products can be used for a variety of downstream applications including transformation, PCR and rolling circle amplification (RCA). The GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells, is a complete kit that includes master mix, positive control, water, and One Shot TOP10 chemically competent E. coli.

Features of the GeneArt Gibson Assembly HiFi Cloning Kit include:
Simple—seamlessly assemble and clone up to six DNA fragments in a single reaction
Efficient—high fidelity provides more correct clones than other methods
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of multiple fragments
GeneArt Gibson Assembly HiFi cloning is a simple, one-step process whereby up to six fragments are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After a 15–60 minute incubation, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones that are ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly HiFi method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, improving the success of finding the right clone, particularly for more challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly HiFi method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly HiFi kits are available in multiple formats, including:
• GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells) (this page)
GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells)
GeneArt Gibson Assembly HiFi Master Mix kits (cloning kit without the cells)

RevertAid H Minus First Strand cDNA Synthesis Kit Thermo Scientific™

Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit includes RevertAid H Minus Reverse Transcriptase, which has a point mutation that completely eliminates RNase H activity. Therefore, it does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA.

Highlights

• High yields of full-length first strand cDNA up to 13 kb
• Increased reaction temperatures in the range of 42 to 55°C
• Supplied with the recombinant RiboLock RNase Inhibitor
• Complete kit—oligo(dT)18 and random hexamer primers included with the kit

Applications

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

Additional Features

The recombinant RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. It is fully compatible with the reverse transcription reaction, as it maintains activity at temperatures up to 55°C.The 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 poly(A), therefore, they can be used for transcription of the 5’-end regions of mRNA or for cDNA synthesis using RNA without the poly(A) tail, e.g., microRNAs. Gene-specific primers may also be used with the kit. The first strand cDNA can be directly used as a template in PCR, realtime PCR or in second strand cDNA synthesis.

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)

GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR 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 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 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).

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.

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)

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 single-tube format speeds up the synthesis procedure and maximizes cDNA recovery. The kit contains pre-mixed components to reduce the number of pipetting steps necessary to complete the procedure.

Features of the Maxima H Minus Double-Stranded cDNA Synthesis Kit include:
• Efficient synthesis of full-length double-stranded cDNA
• Fast—procedure completed in less than two hours
• Convenient—pre-mixed 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

SuperScript™ III CellsDirect™ cDNA Synthesis Kit Invitrogen™

The SuperScript® III CellsDirect cDNA Synthesis Kit is optimized for synthesizing first-strand cDNA directly from a mammalian cell lysate without first isolating the RNA. Lysis and reverse transcription are performed in the same tube, and the resulting first-strand cDNA is ready to use in cloning and PCR. For real-time quantitative RT-PCR, see the note below. The kit has the following advantages:

• Compatible with a wide range of mammalian cell types grown under different treatment conditions
• Single-tube format minimizes reagent loss, sample loss, and handling time
• Total lysate volume is used in first-strand cDNA synthesis reaction, providing greater yields with a limited number of cells and allowing for detection of rare transcripts
• SuperScript® III Reverse Transcriptase, with reduced RNase H activity and higher thermal stability, produces high yields of cDNA in the first-strand synthesis reaction, for greater sensitivity and enhanced detection of rare transcripts
• Generates high-quality cDNA for use in a variety of applications, including cloning and PCR
• Simple protocol takes less than 2 hours

How it works
In traditional RT-PCR, RNA is first isolated from cells in a time-consuming procedure that can lead to a loss of material. Using the SuperScript® III CellsDirect cDNA Synthesis System, the cells are lysed and the cDNA is generated from the lysate in a single tube with minimal handling and no sample loss. DNase I is added to eliminate genomic DNA prior to first-strand synthesis. This kit has been optimized for small cell samples, ranging from 10,000 cells down to a single cell (as measured by serial dilution). The use of SuperScript® III Reverse Transcriptase ensures high specificity and high yields of cDNA from small amounts of starting material—as little as 10 pg total RNA. After synthesis, the first-strand cDNA can be amplified with specific primers by PCR without intermediate organic extractions or ethanol precipitations.

GeneArt™ Gibson Assembly HiFi Cloning Kit, electrocompetent cells Invitrogen™

The GeneArt Gibson Assembly HiFi Cloning Kit enables DNA assembly and cloning via a technique that allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 15–60 minute isothermal reaction. DNA fragments of different lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility of this approach is suitable for small and large DNA constructs and includes both single and multiple inserts. The resulting products can be used for a variety of downstream applications including transformation, PCR and rolling circle amplification (RCA). The GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells, is a complete kit that includes master mix, positive control, water, and ElectroMAX DH10B electrocompetent E. coli.

Features of the GeneArt Gibson Assembly HiFi Cloning Kit include:
Simple—seamlessly assemble and clone up to six DNA fragments in a single reaction
Efficient—high fidelity provides more correct clones than other methods
Flexible—design guidelines allow assembly into any vector of your choice
Convenient—available as master mix kits and cloning kits complete with chemical or electrocompetent cells
Trusted—over 4000 citations in the scientific literature highlighting great success

Seamless assembly of multiple fragments
GeneArt Gibson Assembly HiFi cloning is a simple, one-step process whereby up to six fragments are combined in a proprietary enzymatic mix in order to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). After a 15–60 minute incubation, a portion of the assembly reaction is then transformed into chemically competent or electrocompetent cells, yielding clones that are ready for analysis the next day. The required 20- to 40-base pair end homology is designed into the de novo fragment for synthesis or can be easily engineered by PCR amplification with custom DNA oligos. This special enzyme mix creates a seamless and covalently bound DNA construct providing high efficiency.

Robust method for maximum efficiency
Due to the covalently bound final product, the GeneArt Gibson Assembly HiFi method allows the utilization of chemically competent cells or electrocompetent cells for the highest transformation efficiency, improving the success of finding the right clone, particularly for more challenging constructs. There is no need for restriction enzymes, ligation, or recombination sites, and the method provides a perfectly seamless construct without unwanted extra bases.

Provides great versatility
The GeneArt Gibson Assembly HiFi method provides versatility, can streamline many techniques through the rapid combination, addition, deletion, or exchange of DNA segments, and eliminates the need to subclone, saving time and effort in the cloning workflow.

Convenient formats
GeneArt Gibson Assembly HiFi kits are available in multiple formats, including:
• GeneArt Gibson Assembly HiFi Cloning Kit, electrocompetent cells (with ElectroMAX DH10B electrocompetent cells) (this page)
GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells (with One Shot TOP10 chemically competent cells)
GeneArt Gibson Assembly HiFi Master Mix kits (cloning kit without the cells)

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
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