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Thermo Scientific Cloning Workflow
The Thermo Scientific molecular biology portfolio offers the most reliable genetic content, superior enzymes and advanced PCR tools to support and facilitate your entire cloning workflow. Our products meet the industry's highest quality control standards and provide performance, accuracy and consistency. Find out more about Thermo Scientific products for each of your cloning workflow steps starting from sample preparation to fragment amplification, DNA modification, electrophoresis, and ligation by clicking on the navigation buttons on the menu bar below.
High yields & exceptional value
Thermo Scientific GeneJET nucleic acid purification kits are designed for rapid, efficient, and convenient purification of DNA and RNA from a wide range of samples. The kits utilize a proprietary silica-based membrane technology in the form of a convenient spin-column eliminating the need for expensive resins, toxic phenol-chloroform extractions, or time-consuming alcohol precipitation. Purified DNA or RNA is ready to use in all common molecular biology procedures.
Features
- Efficient nucleic acid extraction and high yields
- High purity of isolated DNA or RNA
- Simple and fast isolation procedure
- Convenient silica-based spin column format
Applications
- Plasmid DNA isolation
- Genomic DNA isolation
- Total RNA isolation
- PCR purification
- Gel extraction
Learn more
Plasmid DNA Purification
- GeneJET Plasmid Miniprep Kit
- GeneJET Plasmid Midiprep Kit
- GeneJET Plasmid Maxiprep Kit
- GeneJET Endo-Free Plasmid Maxiprep Kit
Purification of DNA Fragments
Genomic DNA Purification
- GeneJET Genomic DNA Purification Kit
- GeneJET Plant Genomic DNA Purification Mini Kit
- GeneJET Whole Blood Genomic DNA Purification Mini Kit
- GeneJET FFPE DNA Purification Kit
Total RNA Purification
- GeneJET RNA Purification Kit
- GeneJET Plant RNA Purification Mini Kit
- GeneJET Whole Blood RNA Purification Mini Kit
- GeneJET Stabilized and Fresh Whole Blood RNA Kit
DNA & RNA Cleanup and Concentration
High-fidelity PCR
In cloning applications, it is crucial to maintain accurate DNA sequences during amplification. Thermo Scientific Phusion High-Fidelity DNA Polymerases have extremely low error rates and set a gold standard for high-fidelity PCR.
- Extreme fidelity (error rate 4.4 x 10-7 in Phusion HF Buffer)
- Polymerase speed allows for short extension times (15-30 s/kb)
- Robust performance, minimal optimization needed
- Increased product yields with minimal amounts of enzyme
- Green Buffer for direct loading of PCR products on gels
Reverse Transcriptases
Thermo Scientific Maxima Reverse Transcriptases have been developed through in vitro evolution of M-MuLV reverse transcriptase to yield dramatically improved thermostability, robustness and increased synthesis rate compared to wild-type M-MuLV RT.
Thermal Cyclers
Small and fast, the Thermo Scientific Piko Thermal Cycler delivers superior thermal performance in a compact package and enables PCR runs in as little as 15 minutes. For routine PCR, the Thermo Scientific Arktik Thermal Cycler is a reliable and easy-to-use instrument designed to meet everyday requirements in any PCR laboratory.
Consumables
Thermo Scientific PCR plastics are manufactured with industry-leading processes and extensive quality control to ensure maximum PCR performance. The UTW® (ultra-thin wall) PCR plastics allow shorter PCR run times by improving heat transfer.
Learn more
Restriction digestion as easy as it gets
Thermo Scientific FastDigest Enzymes are an advanced line of 176 restriction enzymes designed for rapid DNA digestion. All FastDigest enzymes are 100% active in the universal FastDigest and FastDigest Green reaction buffers and are able to digest DNA in 5-15 minutes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. As an added convenience, the FastDigest Green buffer allows for direct loading of the reaction mixture on gels.
- 100% activity of all FastDigest enzymes in one buffer
- Complete DNA digestion in 5-15 minutes
- 100% buffer compatibility with downstream applications
- Direct loading of reaction products on gels
- 176 FastDigest Enzymes available
| Old Standard Conventional restriction enzymes | New Standard FastDigest restriction enzymes | |
|---|---|---|
| Buffer system | Up to 20 buffers | One universal buffer |
| Buffer change between enzymatic steps | Frequently necessary | Not necessary |
| Double/multiple restriction digestion | Limited to buffer incompatibility | Any digestion combination possible |
| Reaction time | 1 hour - overnight | 5 - 15 minutes |
| Direct loading on gels | No | Yes |
| Downstream applications | Partial compatibility | 100% compatibility |
| Star activity | Dependent on enzyme properties and reaction conditions | No star activity due to short incubation time |
| Activity definition | 1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme | 1 µl of FastDigest enzyme cleaves 1 µg of substrate DNA in 5 or 15 minutes of FastDigest buffer |
Modification of DNA fragment ends prior to ligation is an essential step to the overall success of a cloning experiment. With the Thermo Scientific FastAP Alkaline Phosphatase and Thermo Scientific Fast DNA End Repair Kit, modification of DNA ends is fast and effective.
FastAP Alkaline Phosphatase
FastAP Alkaline Phosphatase is ideal for rapid and efficient dephosphorylation of linearized vector DNA in just 10 min. Complete thermal inactivation of the enzyme in just 5 minutes makes FastAP a faster and more efficient alternative to SAP.
Features
- Fast 10 min dephosphorylation of all types of DNA ends
- Rapid thermal inactivation with only 5 min at 75°C
- Simultaneous digestion and dephosphorylation of vector DNA
- 100% active in restriction enzyme and PCR buffers
Fast DNA End Repair Kit
The new Fast DNA End Repair Kit is designed for fast and efficient DNA fragment blunting and phosphorylation prior to ligation. This kit can be used with all types of double-stranded DNA:
- Fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated)
- Restriction enzyme digested plasmid DNA
- Double-stranded cDNA
- PCR products with 3'-dA overhangs
Features
- Efficient—blunting and phosphorylation of double-stranded DNA
- Fast—reaction is complete in 5 minutes
- Convenient—reaction components are premixed to reduce pipetting steps
Efficient ligation of DNA fragments following end repair with the Fast DNA End Repair kit (Figure 1). A 130 bp PCR product with 3'- dA overhangs was end-repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier's recommendations and subsequently ligated with T4 DNA Ligase (#EL0014). Ligation conditions: 200 ng of a 130 bp PCR product ligated with 5U T4 DNA Ligase in 1X T4 DNA Ligase Buffer with 5% PEG 4000 for 1 hour at 22°C.
Figure 1. Fast DNA End Repair kit.
Lane M – GeneRuler 1 kb Plus DNA Ladder (#SM1333),
Lane C1 – PCR product,
Lane C2 – PCR product after ligation,
Lane T – PCR product after end repair using Fast DNA End Repair Kit (20°C, 5 min) and ligation,
Lane A – PCR product after end repair using Vendor A kit (20°C, 30 min) and ligation,
Lane B – PCR product after end repair using Vendor B kit (RT, 45 min; 70°C, 10 min) and ligation.
Designed for perfect migration
The Thermo Scientific molecular biology portfolio offers the widest selection of DNA ladders ranging from 10 bp to 48.5 kb for accurate analysis of linear double-stranded DNA in agarose or polyacrylamide gels. All of our DNA ladders are composed of individual chromatography-purified DNA fragments produced with a patented technology used to prepare pharmaceutical grade plasmid DNA.
Features
- Broad selection of DNA ladders.
- Composed of individual chromatography-purified DNA fragments.
- Bright, sharp bands.
- Ideal for in-gel DNA quantification.
- Supplied with extra loading dye for sample DNA.
- Ready-to-use DNA ladders are stable at room temperature for at least 6 months.
| DNA Ladder Line | Description | Run Time |
|---|---|---|
| GeneRuler DNA Ladders | 1 kb Ladder, 100 bp ladder and other popular DNA ladders for accurate DNA sizing | 45 min |
| FastRuler DNA Ladders | Rapid DNA separation. Ideal for sizing and quantification of PCR products | 8–14 min |
| MassRuler DNA Ladders | Accurate in-gel quantification of DNA fragments | 45 min |
| O'RangeRuler DNA Ladders | Step-ladders designed for precise sizing of DNA fragments in agarose or non-denaturing polyacrylamide gels | 45–90 min |
| NoLimits Individual DNA Fragments | A collection of 36 highly-purified individual DNA fragments ranging in size from 10–20,000 bp | Varies |
Figure 2. 1.1% TopVision Agarose, 1X TAE, 7 V/cm, 30 min. NoLimits DNA fragments from 300 bp to 10 kb
Figure 3. GeneRuler DNA Ladders
Rapid DNA Ligation Kit
The Rapid DNA Ligation Kit allows sticky-end or blunt-end DNA ligation in only 5 min at room temperature. The kit contains T4 DNA Ligase and a specially formulated 5X Rapid Ligation Buffer.
Features
- Fast—ligation of either sticky- or blunt-end DNA in only 5 minutes.
- Convenient—reaction mixture can be used directly for bacterial transformation.
CloneJET PCR Cloning Kit
The CloneJET PCR Cloning Kit is an advanced positive-selection system for high efficiency cloning of PCR products generated with any thermostable DNA polymerase. Additionally, any other blunt or sticky-end DNA fragment can also be cloned using the CloneJET kit. The kit is compatible with phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 minutes and yields more than 99% recombinant clones.
Features
- Fast—PCR cloning in only 5–10 minutes.
- High efficiency— >99% positive clones.
- No cloning background with the positive selection pJET1.2 vector.
- Economical—eliminates the need for expensive blue/white screening.
Learn more
Figure 4. pJET1.2/blunt vector map.
High yields & exceptional value
Thermo Scientific GeneJET nucleic acid purification kits are designed for rapid, efficient, and convenient purification of DNA and RNA from a wide range of samples. The kits utilize a proprietary silica-based membrane technology in the form of a convenient spin-column eliminating the need for expensive resins, toxic phenol-chloroform extractions, or time-consuming alcohol precipitation. Purified DNA or RNA is ready to use in all common molecular biology procedures.
Features
- Efficient nucleic acid extraction and high yields
- High purity of isolated DNA or RNA
- Simple and fast isolation procedure
- Convenient silica-based spin column format
Applications
- Plasmid DNA isolation
- Genomic DNA isolation
- Total RNA isolation
- PCR purification
- Gel extraction
Learn more
Plasmid DNA Purification
- GeneJET Plasmid Miniprep Kit
- GeneJET Plasmid Midiprep Kit
- GeneJET Plasmid Maxiprep Kit
- GeneJET Endo-Free Plasmid Maxiprep Kit
Purification of DNA Fragments
Genomic DNA Purification
- GeneJET Genomic DNA Purification Kit
- GeneJET Plant Genomic DNA Purification Mini Kit
- GeneJET Whole Blood Genomic DNA Purification Mini Kit
- GeneJET FFPE DNA Purification Kit
Total RNA Purification
- GeneJET RNA Purification Kit
- GeneJET Plant RNA Purification Mini Kit
- GeneJET Whole Blood RNA Purification Mini Kit
- GeneJET Stabilized and Fresh Whole Blood RNA Kit
DNA & RNA Cleanup and Concentration
High-fidelity PCR
In cloning applications, it is crucial to maintain accurate DNA sequences during amplification. Thermo Scientific Phusion High-Fidelity DNA Polymerases have extremely low error rates and set a gold standard for high-fidelity PCR.
- Extreme fidelity (error rate 4.4 x 10-7 in Phusion HF Buffer)
- Polymerase speed allows for short extension times (15-30 s/kb)
- Robust performance, minimal optimization needed
- Increased product yields with minimal amounts of enzyme
- Green Buffer for direct loading of PCR products on gels
Reverse Transcriptases
Thermo Scientific Maxima Reverse Transcriptases have been developed through in vitro evolution of M-MuLV reverse transcriptase to yield dramatically improved thermostability, robustness and increased synthesis rate compared to wild-type M-MuLV RT.
Thermal Cyclers
Small and fast, the Thermo Scientific Piko Thermal Cycler delivers superior thermal performance in a compact package and enables PCR runs in as little as 15 minutes. For routine PCR, the Thermo Scientific Arktik Thermal Cycler is a reliable and easy-to-use instrument designed to meet everyday requirements in any PCR laboratory.
Consumables
Thermo Scientific PCR plastics are manufactured with industry-leading processes and extensive quality control to ensure maximum PCR performance. The UTW® (ultra-thin wall) PCR plastics allow shorter PCR run times by improving heat transfer.
Learn more
Restriction digestion as easy as it gets
Thermo Scientific FastDigest Enzymes are an advanced line of 176 restriction enzymes designed for rapid DNA digestion. All FastDigest enzymes are 100% active in the universal FastDigest and FastDigest Green reaction buffers and are able to digest DNA in 5-15 minutes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. As an added convenience, the FastDigest Green buffer allows for direct loading of the reaction mixture on gels.
- 100% activity of all FastDigest enzymes in one buffer
- Complete DNA digestion in 5-15 minutes
- 100% buffer compatibility with downstream applications
- Direct loading of reaction products on gels
- 176 FastDigest Enzymes available
| Old Standard Conventional restriction enzymes | New Standard FastDigest restriction enzymes | |
|---|---|---|
| Buffer system | Up to 20 buffers | One universal buffer |
| Buffer change between enzymatic steps | Frequently necessary | Not necessary |
| Double/multiple restriction digestion | Limited to buffer incompatibility | Any digestion combination possible |
| Reaction time | 1 hour - overnight | 5 - 15 minutes |
| Direct loading on gels | No | Yes |
| Downstream applications | Partial compatibility | 100% compatibility |
| Star activity | Dependent on enzyme properties and reaction conditions | No star activity due to short incubation time |
| Activity definition | 1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme | 1 µl of FastDigest enzyme cleaves 1 µg of substrate DNA in 5 or 15 minutes of FastDigest buffer |
Modification of DNA fragment ends prior to ligation is an essential step to the overall success of a cloning experiment. With the Thermo Scientific FastAP Alkaline Phosphatase and Thermo Scientific Fast DNA End Repair Kit, modification of DNA ends is fast and effective.
FastAP Alkaline Phosphatase
FastAP Alkaline Phosphatase is ideal for rapid and efficient dephosphorylation of linearized vector DNA in just 10 min. Complete thermal inactivation of the enzyme in just 5 minutes makes FastAP a faster and more efficient alternative to SAP.
Features
- Fast 10 min dephosphorylation of all types of DNA ends
- Rapid thermal inactivation with only 5 min at 75°C
- Simultaneous digestion and dephosphorylation of vector DNA
- 100% active in restriction enzyme and PCR buffers
Fast DNA End Repair Kit
The new Fast DNA End Repair Kit is designed for fast and efficient DNA fragment blunting and phosphorylation prior to ligation. This kit can be used with all types of double-stranded DNA:
- Fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated)
- Restriction enzyme digested plasmid DNA
- Double-stranded cDNA
- PCR products with 3'-dA overhangs
Features
- Efficient—blunting and phosphorylation of double-stranded DNA
- Fast—reaction is complete in 5 minutes
- Convenient—reaction components are premixed to reduce pipetting steps
Efficient ligation of DNA fragments following end repair with the Fast DNA End Repair kit (Figure 1). A 130 bp PCR product with 3'- dA overhangs was end-repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier's recommendations and subsequently ligated with T4 DNA Ligase (#EL0014). Ligation conditions: 200 ng of a 130 bp PCR product ligated with 5U T4 DNA Ligase in 1X T4 DNA Ligase Buffer with 5% PEG 4000 for 1 hour at 22°C.
Figure 1. Fast DNA End Repair kit.
Lane M – GeneRuler 1 kb Plus DNA Ladder (#SM1333),
Lane C1 – PCR product,
Lane C2 – PCR product after ligation,
Lane T – PCR product after end repair using Fast DNA End Repair Kit (20°C, 5 min) and ligation,
Lane A – PCR product after end repair using Vendor A kit (20°C, 30 min) and ligation,
Lane B – PCR product after end repair using Vendor B kit (RT, 45 min; 70°C, 10 min) and ligation.
Designed for perfect migration
The Thermo Scientific molecular biology portfolio offers the widest selection of DNA ladders ranging from 10 bp to 48.5 kb for accurate analysis of linear double-stranded DNA in agarose or polyacrylamide gels. All of our DNA ladders are composed of individual chromatography-purified DNA fragments produced with a patented technology used to prepare pharmaceutical grade plasmid DNA.
Features
- Broad selection of DNA ladders.
- Composed of individual chromatography-purified DNA fragments.
- Bright, sharp bands.
- Ideal for in-gel DNA quantification.
- Supplied with extra loading dye for sample DNA.
- Ready-to-use DNA ladders are stable at room temperature for at least 6 months.
| DNA Ladder Line | Description | Run Time |
|---|---|---|
| GeneRuler DNA Ladders | 1 kb Ladder, 100 bp ladder and other popular DNA ladders for accurate DNA sizing | 45 min |
| FastRuler DNA Ladders | Rapid DNA separation. Ideal for sizing and quantification of PCR products | 8–14 min |
| MassRuler DNA Ladders | Accurate in-gel quantification of DNA fragments | 45 min |
| O'RangeRuler DNA Ladders | Step-ladders designed for precise sizing of DNA fragments in agarose or non-denaturing polyacrylamide gels | 45–90 min |
| NoLimits Individual DNA Fragments | A collection of 36 highly-purified individual DNA fragments ranging in size from 10–20,000 bp | Varies |
Figure 2. 1.1% TopVision Agarose, 1X TAE, 7 V/cm, 30 min. NoLimits DNA fragments from 300 bp to 10 kb
Figure 3. GeneRuler DNA Ladders
Rapid DNA Ligation Kit
The Rapid DNA Ligation Kit allows sticky-end or blunt-end DNA ligation in only 5 min at room temperature. The kit contains T4 DNA Ligase and a specially formulated 5X Rapid Ligation Buffer.
Features
- Fast—ligation of either sticky- or blunt-end DNA in only 5 minutes.
- Convenient—reaction mixture can be used directly for bacterial transformation.
CloneJET PCR Cloning Kit
The CloneJET PCR Cloning Kit is an advanced positive-selection system for high efficiency cloning of PCR products generated with any thermostable DNA polymerase. Additionally, any other blunt or sticky-end DNA fragment can also be cloned using the CloneJET kit. The kit is compatible with phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 minutes and yields more than 99% recombinant clones.
Features
- Fast—PCR cloning in only 5–10 minutes.
- High efficiency— >99% positive clones.
- No cloning background with the positive selection pJET1.2 vector.
- Economical—eliminates the need for expensive blue/white screening.
Learn more
Figure 4. pJET1.2/blunt vector map.