Build Better Molecular Biology Workflows

Prepare for discovery Molecular biology workflow solutions Molecular biology Unlocking scientific discovery: making molecular biology accessible to all From the beginning, our scientists have set out to commercialize tools to help advance molecular biology. In the early 1990s, Invitrogen™ SuperScript™ Reverse Transcriptase and TOPO™ cloning technology were introduced to the market. TOPO cloning technology was followed by many other products to speed up and simplify previously complex techniques. Over the decades, we have continued to develop new products and improve old favorites. Helping our customers apply these tools to solve new research challenges is our purpose. Here we provide foundational information and introduce the latest tools to aid your research. Find additional information at thermofisher.com/molecularmasterpiece. Contents Sample preparation Tools and methods 7 Automation platforms 10 Automation-compatible kits 11 DNA and RNA isolation 14 Reverse transcription Considerations 17 Reagent selection 18 Genomic DNA removal 21 Primers 22 PCR Thermal cycler considerations 25 Plastics essentials 28 PCR enzymes 32 Oligo design and selection 34 Electrophoresis Workflow 38 E-Gel product selection 41 Electrophoresis reagents 42 Cloning Technologies overview 45 Restriction enzyme cloning 46 PCR cloning 48 Cloning with synthetic DNA 50 Transformation 51 Isothermal amplification Overview 55 LAMP 56 MDA-WGA 58 RCA 59 RPA 60 Resources Educational resources 63 Mobile apps 64 Custom Commercial Supply 64 FAQs 65 Ordering information 67 Sample preparation Table of contents 5 Nucleic acid isolation is a crucial first step in the molecular biology workflow, whether you are isolating genomic DNA (gDNA) or RNA. Selecting nucleic acid purification products that are optimized to help provide maximum yield, purity, and integrity from virtually any sample type and application is important for your research success. Sample preparation Advance your research at thermofisher.com/kingfisher 6 Table of contents Portfolio of solutions for your nucleic acid isolation DNA type For gDNA extraction, cfDNA isolation, plasmid isolation, and DNA cleanup RNA type For purification of total RNA, cell-free RNA (cfRNA), transcriptome RNA, messenger RNA (mRNA), microRNA (miRNA) and other small RNA, and sequence-specific RNA capture RNA Sequence-specific RNA Viral RNA Purified and small RNA MicroRNA (cfRNA) Cell-free RNA (RNA + DNA) Total nucleic acid mRNA Total RNA DNA Sequence-specific Oligonucleotide Nucleic acid Agarose gel cleanup PCR product (cfDNA) Cell-free DNA Microbiome Genomic Plasmid Learn more at thermofisher.com/kingfisherkits Sample preparation Table of contents 7 Understanding common nucleic acid isolation methods Magnetic beads: 0.5–1.0 μm particles with a paramagnetic core and modified shell (e.g., Applied Biosystems™ MagMAX™ kits and Invitrogen™ Dynabeads™ magnetic beads) Samples are lysed in solution and allowed to bind nucleic acid to magnetic particles based on specific surface modifications. Application of an external magnetic field rapidly collects the particles. Rounds of release, wash, and recapture enable purification of the desired nucleic acid. Automated purification instruments: automated processing of magnetic particles in a microplate format (e.g., Thermo Scientific™ KingFisher™ purification systems) Samples are processed by moving magnetic beads (not liquid). The system utilizes magnetic rods covered with a disposable, specially designed tip comb and plates. The instrument functions without any dispensing or aspiration parts or devices. Before the run, samples and reagents, including magnetic particles, are dispensed into plates according to default protocols that are installed on the instrument. Benefits: • No risk of clogging • Increased target capture efficiency • Rapid collection and concentration of sample • Specialized equipment not required • Scalability Benefits: • Process 6–96 samples per run • 24- or 96-well plates for different input volumes • Easily edit, modify, or create new protocols • All the benefits of magnetic beads (below) Learn more at thermofisher.com/sampleprep Helpful tip Invitrogen™ Dynabeads™ magnetic beads are suitable for a variety of applications in addition to nucleic acid purification. Visit thermofisher.com/dynabeads to learn about additional applications, including cell isolation, immunoprecipitation, and chromatin immunoprecipitation (ChIP). Sample preparation 8 Table of contents Spin columns: Glass fiber, derivatized silica, or ion exchange membrane in column (e.g., Thermo Scientific™ GeneJET™ and Invitrogen™ PureLink™ kits) Samples are lysed and passed through the membrane using centrifugal or vacuum force. Wash and elution solutions are subsequently passed through the membrane, and the sample is collected into a tube by centrifugation. Benefits: • Convenience • Ease of use • Throughput flexibility • Specialized equipment not required Organic extraction: Phenol-chloroform solution (e.g., Invitrogen™ DNAzol™ and TRIzol™ Reagents) After homogenizing the sample with TRIzol Reagent, chloroform is added and the mixture separates into a clear upper aqueous layer containing RNA, an interphase layer, and a pink lower organic layer containing the DNA and protein. RNA is precipitated from the upper aqueous layer with isopropanol. DNA is precipitated from the interphase and organic layers with ethanol. Protein is precipitated from the phenol–ethanol supernatant with isopropanol. Benefits: • Efficient lysis of cells and tissue • Rapid denaturation of nucleases • Stabilization of nucleic acids • Great for fatty and cartilaginous samples Learn more at thermofisher.com/sampleprep Sample preparation Table of contents 9 Automation platforms: Find a model that meets your needs Optimize and automate your DNA and RNA, cell, and protein purification workflows with KingFisher systems. When used with compatible bead-based reagents such as MagMAX and Dynabeads products, these versatile instruments enable the automation of DNA, RNA, protein, and cell isolation procedures. Learn more at thermofisher.com/kingfisher KingFisher instrument: Duo Prime Flex Apex Presto PlasmidPro Instrument size Compact benchtop Benchtop Benchtop Benchtop—integrates with robotic liquid handler Benchtop Throughput level Low to medium High High Ultrahigh Low Processing volume range • 50–1,000 μL: 12-pin magnet head • 200–5,000 μL: 6-pin magnet head • 20–100 μL: skirted PCR plate • 20–200 μL: 96-well plate • 50–1,000 μL: 96 deep-well plate • 200–5,000 μL: 24 deep-well plate • 15–1,000 μL: 96 deep-well plate • 15–200 μL: 96-well KingFisher standard plate • 10–80 μL: 96-well PCR plate • 30–5,000 μL: 24 deep-well plate • 30–200 μL: 96 storage tubes • 200–1,000 μL: 24 storage tubes • 50–1,000 μL: 96 deep-well plate • 200–5,000 μL: 24 deep-well plate • 50–150 μL: KingFisher 96 plate • 150 mL overnight bacterial culture up to 6 OD or 106 cells/mL in LB medium Samples per run 6 or 12 24 or 96 24 or 96 24 or 96 One Customizable protocols Yes Yes Yes, with touchscreen or PC software Yes No Heating and cooling • 10°C to 75°C (plate row block A) • 4°C to 75°C (elution strip block) • From 5°C above ambient temperature to 115°C • From 4°C above ambient temperature to 100°C • Cooling down to 4°C • From 5°C above ambient temperature to 115°C NA Ultraviolet lamp 8 W (up to 16 hr) No 2 UV lamps, max 23 hr 59 min No No Additional details For Research Use Only. Not for use in diagnostic procedures. For Laboratory Use For Laboratory Use For Laboratory Use For Research Use Only. Not for use in diagnostic procedures. Designed for automated endotoxin-free maxi preps of plasmid DNA. Prep plates Select program Load plates Press start Run time ** Can vary depending on application and instrument. 25–120 min** <15 min** 1 min 1 min Typical run with a KingFisher instrument Resource Use our selection tool to find the right magnetic bead–based kit for your automated sample preparation. Find out more at thermofisher.com/kingfisherkits The graphic shows the expected run time for sample preparation when using a KingFisher instrument and prefilled plates. It takes less than 15 minutes to prep plates, 1 minute to select the program or protocol, and 1 minute to load plates. You can then press start and walk away for 25–120 minutes, depending on the sample and analyte type. The Thermo Scientific™ KingFisher™ PlasmidPro™ Maxi Processor uses a cartridgebased system to automate the preparation of endotoxin-free plasmid suitable for transfection. Learn more and request a demo at thermofisher.com/kingfisher. Sample preparation 10 Table of contents Selecting the right DNA and RNA isolation kits for your downstream research Application Automation-ready extraction kit and reagents Cat. No. Cancer research MagMAX Cell-Free DNA Isolation Kit A29319 MagMAX Cell-Free Total Nucleic Acid Isolation Kit A36716 MagMAX mirVana Total RNA Isolation Kit A27828 MagMAX FFPE DNA/RNA Ultra Kit A31881 MagMAX DNA Multi-Sample Ultra 2.0 Kit A36570 MagMAX Sequential DNA/RNA Kit A65309 Dynabeads FlowComp Human CD3 Kit 11365D Dynabeads FlowComp Human CD4 Kit 11361D Dynabeads FlowComp Human CD8 Kit 11362D Dynabeads Untouched Human T Cells 11344D Dynabeads Untouched Human CD4 T Cells Kit 11346D Dynabeads CD15* 11137D Dynabeads CD4 11145D Dynabeads Epithelial Enrich 16102 Dynabeads MyOne CD45 Leukocyte Depletion 11170D * Available in 2 mL sizes with automation scripts. Learn more about cell isolation products. Application Automation-ready extraction kit and reagents Cat. No. Genomics MagMAX DNA Multi-Sample Ultra 2.0 Kit A36570 MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit A48383R MagMAX mirVana Total RNA Isolation Kit A27828 NGS Dynabeads Streptavidin for Target Enrichment 65606D MagMAX Pure Bind Beads A58521 Go directly to our kit selection tool at thermofisher.com/kingfisherkits Cancer research: Applied Biosystems™ MagMAX™ cell-free nucleic acid kits on KingFisher instruments are excellent for liquid biopsy research. They are optimized specifically for enrichment of cfDNA and cell-free total nucleic acid (cfTNA), enabling highly reproducible recovery, flexible input volumes, and efficient protocols. Learn more about sample preparation solutions for liquid biopsy. DNA research: Using the Applied Biosystems™ MagMAX™ DNA Multi-Sample Ultra 2.0 Kit and KingFisher instruments, you can isolate gDNA from 50 μL to 2 mL of whole blood, saliva, buffy coat, buccal swabs, or other biological samples. The resulting purified gDNA is ideal for many downstream molecular biology applications such as real-time PCR (qPCR), next-generation sequencing (NGS), microarray analysis, and other applications. Sample preparation Table of contents 11 Selecting the right DNA isolation kits for your downstream research Application Automation-ready extraction kit and reagents Cat. No. Infectious disease research MagMAX Viral/Pathogen Nucleic Acid Isolation Kit for HIV-1 dried blood spots A53770 MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit A48383R MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, with bead tubes A42358 MagMAX DNA Multi-Sample Ultra 2.0 Kit A36570 MagMAX Prime Viral/Pathogen NA Isolation Kit A58145 Dynabeads Intact Virus Enrichment 10700D Application Technologies for plasmid DNA isolation Cat. No. Plasmid purification GeneJET Plasmid Miniprep Kit K0502 GeneJET Endo-Free Plasmid Maxiprep Kit K0861 PureLink Fast Low-Endotoxin Midi Plasmid Purification Kit A35892 PureLink HiPure Plasmid Filter Maxiprep Kit K210016 PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit A33073 MagMAX Pro HT NoSpin Plasmid Miniprep Kit A58309 KingFisher PlasmidPro Maxi Processor Endotoxin-Free Cartridge A54072 Go directly to our kit selection tool at thermofisher.com/kingfisherkits Infectious disease research: Applied Biosystems™ MagMAX™ viral/pathogen kits on KingFisher instruments provide a sensitive and simple method for nucleic acid extraction from samples containing viruses or other pathogens. Learn more at thermofisher.com/mvpprime. Plasmid purification: Choose from our wide range of high-performing, cost-effective Thermo Scientific™ and Invitrogen™ kits for plasmid DNA isolation designed to isolate plasmid DNA at the purity and scale you need. Learn more at thermofisher.com/plasmid. Sample preparation Helpful tip Select the best plasmid preparation kit for your application with the Plasmid Purification Selection Tool. 12 Table of contents Selecting the right DNA isolation kits for your downstream research Application Automation-ready extraction kit and reagents Cat. No. Surveillance MagMAX Wastewater Ultra Nucleic Acid Isolation Kit with Virus Enrichment A52610 MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit A48383R MagMAX mirVana Total RNA Isolation Kit A27828 MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, with bead tubes A42358 MagMAX Prime Viral/Pathogen NA Isolation Kit A58145 Application Automation-ready extraction kit and reagents Cat. No. IP and co-IP Dynabeads Streptavidin for Target Enrichment 65606D Dynabeads M-270 Streptavidin 65305 Dynabeads M-280 Streptavidin 11205D Dynabeads MyOne Streptavidin T1 65601 Dynabeads MyOne Streptavidin C1 65001 Wastewater DNA and RNA for disease surveillance: Applied Biosystems™ MagMAX™ wastewater kits offer an efficient and simple method for extracting high-quality nucleic acids from wastewater, sewage, or sludge samples for disease surveillance workflows. The purified DNA and RNA are excellent for use in a variety of downstream applications such as quantitative PCR, digital PCR, and NGS. Streptavidin: Utilization of Invitrogen™ Dynabeads™ streptavidin magnetic beads offers an exceptional balance of capacity and yield, reproducibility, purity, and cost for smaller-scale isolation of specific proteins (e.g., immunoprecipitation (IP)) and protein complexes (co-immunoprecipitation (co-IP)) and other applications. Go directly to our kit selection tool at thermofisher.com/kingfisherkits Sample preparation Table of contents 13 Helpful tip If you are not ready to process your RNA sample, simply store it in Invitrogen™ RNAlater™ Stabilization Solution for use at a later time. Visit thermofisher.com/stabilizerna. Selecting the right DNA and RNA isolation kits For the quality and performance you need, a full suite of products for DNA and RNA isolation is available for a wide range of sample types, throughputs, and input quantities. To use our online kit selection guide, go to thermofisher.com/rnaselection. Learn more at thermofisher.com/rnapreps Applied Biosystems™ and Invitrogen™ technologies for DNA and total RNA isolation Capabilities Process a large amount of tissue Fast isolation of RNA from a variety of samples High-throughput purification of RNA and DNA Process cells for gene expression Kits TRIzol reagents PureLink kits MagMAX kits Cells-to-CT kits Prep time 30–60 min <20 min 45 min ≤10 min Sample types Most samples, particularly those more difficult to lyse Bacteria, liquid, blood, cells, yeast, plants, tissue Blood, plants, saliva, urine, stool, soil, plasma, serum* Cultured cells Starting material 100 mg of tissue or 107 cells 108 cells, 200 mg of animal tissue, 250 mg of plant tissue, 0.2 mL of blood, 5 x 108 yeast, 109 bacteria Variable depending on sample 1–100,000 cells Yield 106 epithelial cells: 8–15 μg; 100 mg tobacco leaf: 73 μg (variable depending on sample) Up to 350 μg Variable depending on sample NA High throughput–compatible No Yes Yes Yes Technology Organic extraction Silica membrane spin column/filter plate Magnetic beads Crude lysate * Specialty kits with optimized chemistry are available for extraction of cell-free DNA/RNA, total RNA, gDNA, and total nucleic acid. Sample preparation 14 Table of contents Reverse transcription Table of contents 15 Find technical resources on reverse transcription at thermofisher.com/rteducation Reverse transcription is the synthesis of complementary DNA (cDNA) from an RNA template using reverse transcriptase enzymes. The cDNA can be used as a template for PCR amplification, cDNA library construction, RNA sequencing, and more. Selecting the right reverse transcriptase is critical to detecting low-abundance RNAs in a sample and obtaining high yields of full-length cDNA. 16 Table of contents Considerations for selecting the right reverse transcriptase 9.0 6.0 3.0 1.5 0.5 23.1 kb 9.4 6.6 2.3 2 0.01 U/uL Heparin 0.05% Bile salts No inhibitors 10% isopropanol 5% BugBuster 0.0025% Formalin SSIII SSIII SSIII SSIII SSIII SSIII SuperScript III enzyme SSIV SuperScript IV enzyme SSIV SSIV SSIV SSIV SSIV P P P P T T T T BR BR BR BL BL BL Q Q Q Q N N N N N † DNA/Hind III SSIV P T 8.0 kb 6.0 4.0 3.0 2.0 1.5 1.0 0.5 kb N Sensitivity, thermostability, processivity, and inhibitor tolerance of reverse transcriptases all affect the quantity and length of cDNA synthesized. Sensitivity The ability of a reverse transcriptase to generate cDNA from the least amount of input RNA is an important attribute when working with low-copy genes or difficult sample sources where RNA may have already degraded. Thermostability Thermostable reverse transcriptases allow reactions to occur at higher temperatures, which help denature RNA with complex secondary structures or high GC content, for generation of longer cDNA, higher cDNA yields, and better coverage of RNA populations in the cDNA. Processivity Processivity is the enzyme's ability to add consecutive nucleotides without releasing the template. Highly processive reverse transcriptases allow synthesis of longer cDNA strands in a shorter reaction time, and overall better efficiency in making full-length cDNA. Inhibitor tolerance Compounds that have inhibitory effects on reverse transcriptases are common in RNA samples even after purification. Their sources include reagents used for RNA isolation and contaminants carried over from biological samples. Reverse transcriptases resistant to common inhibitors help minimize inconsistent or suboptimal results in cDNA-based assays. 30 M hematin SuperScript III enzyme SuperScript IV enzyme P N T Q SuperScript III enzyme SuperScript IV enzyme P N T Q SuperScript III enzyme SuperScript IV enzyme P N T Q 0.01 U/L heparin 0.5% bile salts 8.0 kb 6.0 4.0 3.0 2.0 1.0 1.5 0.5 SuperScript III enzyme SuperScript IV enzyme P BR Q BL N log10[degraded RNA] (ng) Ct 40 38 36 34 32 30 28 26 0 0.5 1.0 1.5 2.0 SuperScript III enzyme SuperScript IV enzyme Q T P BL N BR Time (min) Activity (%) 120 100 80 60 40 20 0 0 10 20 30 40 50 60 A C B cDNA synthesis in 10 min D Degraded Arabidopsis RNA Target: glutamine synthetase cDNA synthesis at 50°C cDNA synthesis in the presence of common inhibitors (A) Sensitivity, (B) thermostability, (C) processivity, and (D) inhibitor tolerance of reverse transcriptases can affect the quantity and length of cDNA. P, BR, Q, BL, T, and N represent enzymes from other suppliers. Learn more at thermofisher.com/reverse-transcription Reverse transcription Table of contents 17 Product format Stand-alone enzyme First-strand cDNA synthesis kit First-strand cDNA synthesis master mix for RT-qPCR Recommended product Invitrogen™ SuperScript™ IV Reverse Transcriptase Invitrogen™ SuperScript™ IV First-Strand Synthesis System Invitrogen™ SuperScript™ IV VILO™ Master Mix Applications RT-PCR, RT-qPCR, cloning, cDNA library construction, RACE, RNA-Seq RT-PCR, RT-qPCR, cloning, cDNA library construction, RACE, RNA-Seq RT-qPCR Total RNA input 1 pg–5 μg 1 pg–5 μg 0.01 pg–2.5 μg Optimal reaction temperature 50–55°C 50–55°C 50–55°C Reverse transcription time 10 min 10 min 10 min High cDNA yield with challenging or degraded RNA • • • Includes PCR step Available formats • Stand-alone enzyme • cDNA synthesis kit • cDNA synthesis kit with ezDNase enzyme • SuperScript IV VILO Master Mix • SuperScript IV VILO Master Mix with ezDNase enzyme Reverse transcription reagent selection guide Most convenient and fewest pipetting steps for RT-qPCR applications Complete cDNA synthesis kit with all reaction components Ability to optimize reaction components and conditions Did you know? These products are free of OPE and NPE, making them safer for aquatic life. Learn more at thermofisher.com/superscript Reverse transcription 18 Table of contents One-step RT-PCR kit Template switching master mix cDNA PreAmp kit Direct RT kit Invitrogen™ SuperScript™ IV UniPrime™ One-Step RT-PCR System Invitrogen™ SuperScript™ IV Template Switching RT Master Mix Invitrogen™ SuperScript™ IV Single Cell/Low Input cDNA PreAmp Kit Invitrogen™ SuperScript™ IV CellsDirect™ cDNA Synthesis Kit RT-PCR RT-PCR, RACE, RNA-Seq RT-qPCR, RNA-Seq RT-PCR, RT-qPCR 0.01 pg–1 μg 1–1,000 cells or 2 pg–10 ng 1–1,000 cells or 2 pg–10 ng 1–10,000 cells 50–55°C 50°C 50°C 50–55°C 10 min 30 min 30 min 10 min • • • • • • SuperScript IV UniPrime One-Step RT-PCR System (colored) • SuperScript IV UniPrime One-Step RT-PCR System (dye-free) • Invitrogen™ SuperScript™ IV Template Switching RT Master Mix • SuperScript IV Single Cell/Low Input cDNA PreAmp Kit • SuperScript IV CellsDirect cDNA Synthesis Kit Go from mammalian cell lysate to cDNA synthesis without isolating RNA Optimized template switching for RACE and RNA-Seq applications cDNA synthesis and amplification directly from intact single cells or low amounts of total RNA Most convenient and fewest pipetting steps for RT-PCR applications Did you know? The standard enzyme format is incompatible with lyophilization because of the glycerol in the storage buffer. The lyo-ready (lyophilization-ready) format of SuperScript reverse transcriptases has a glycerol content below 0.1% and offers greater stability for lyophilized molecular assay kits. Learn more at thermofisher.com/lyoreadyenzymes. Reverse transcription Table of contents 19 Product Invitrogen™ SUPERase•In™ RNase Inhibitor Invitrogen™ RNaseOUT™ T Recombinant Ribonuclease Inhibitor Applied Biosystems™ RNase Inhibitor Invitrogen™ Ambion™ RNase Inhibitor, cloned Mechanism Protein-based inhibitor of nonhuman origin that noncovalently binds to and inhibits the most common and troublesome RNases, including RNase A, B, C, 1, and T1 Noncompetitive inhibitor of pancreatic-type ribonucleases such as RNase A 50 kDa recombinant enzyme used to inhibit RNase activity Recombinant human protein produced in E. coli and inhibitor of neutral pancreatic RNase A–type enzymes Sizes available • 2,500 units • 10,000 units • 5,000 units • 2,000 units • 1,000 units • 2,500 units • 10,000 units RNase inhibitors To ensure your RNA template is protected and reverse transcription is successful, it’s necessary to include RNase inhibitors. This step is crucial for preserving the integrity of the sample and achieving accurate and reproducible results. Note: All SuperScript IV kit formats either contain an RNase inhibitor or are supplied with one. Reverse transcription 20 Table of contents RNA purification methods, including protocols with DNase digestion on column, often fail to completely remove gDNA. Amplification of contaminating gDNA can cause inaccurate results. Moreover, traditional gDNA decontamination protocols with DNase I include time-consuming DNase inactivation steps under conditions that can damage RNA and affect results. Invitrogen™ SuperScript™ IV VILO™ Master Mix is available in a format with the novel dsDNA‑specific Invitrogen™ ezDNase™ Enzyme, which enables efficient, fast, and gentle gDNA removal from RNA samples to help ensure high confidence in RT-PCR and RT-qPCR results. Genomic DNA removal ezDNase treatment (37°C, 2 min) Primer annealing (25°C, 10 min) Reverse transcription (50°C, 10 min) Reverse transcriptase inactivation (85°C, 5 min) ~27 minutes RNA DNase I treatment (37°C, 20 min) Primer annealing (25°C, 10 min) Reverse transcription (42°C, 60 min) Enzyme inactivation (85°C, 5 min) ~105 minutes DNase I inactivation/+EDTA (65°C, 10 min) RNA Traditional cDNA synthesis workflow with DNase I ezDNase treatment (37°C, 2 min) Primer annealing (25°C, 10 min) Reverse transcription (50°C, 10 min) Reverse transcriptase inactivation (85°C, 5 min) ~27 minutes RNA DNase I treatment (37°C, 20 min) Primer annealing (25°C, 10 min) Reverse transcription (42°C, 60 min) Enzyme inactivation (85°C, 5 min) ~105 minutes DNase I inactivation/+EDTA (65°C, 10 min) RNA SuperScript IV VILO Master Mix cDNA synthesis workflow with ezDNase Enzyme Learn more at thermofisher.com/4vilo Reverse transcription Table of contents 21 Two most common primers used in reverse transcription A U C G G G A C C U G A U U C U G A A A A A A 5´ A 5´ 3´ 3´ Poly(A) tall T T T Oligo(dT) primer T T T T A C G G G A C C G A G C C C C G T T A G 5´ T T T A 5´ 3´ 3´ G C T Specific primer C T C A G A A A G G U C A C G A U U U C C C U U A G G 5´ A A A 5´ 3´ 3´ N N N Random primers N N N 3´ 5´ N N N N N N 5´ A U C G G G A C C U G A U U C U G A A A A A A A 5´ 3´ 3´ Poly(A) tall T T T Oligo(dT) primer T T T T A C G G G A C C G A G C C C C G T T A G 5´ T T T A 5´ 3´ 3´ G C T Specific primer C T C A G A A A G G U C A C G A U U U C C C U U A G G 5´ A A A 5´ 3´ 3´ N N N Random primers N N N 3´ 5´ N N N N N N Helpful tip To avoid poly(A) slippage during priming, anchored oligo(dT) primers can be used to anneal to the 5´ end of the poly(A) tail of mRNA and prevent priming within the poly(A) tail. Learn more about selection of primers for reverse transcription at thermofisher.com/rteducation. The priming strategy you choose for reverse transcription is important for cDNA synthesis efficiency, consistency, and yield. Each primer type has its benefits and drawbacks, depending on the individual target RNA. For full-length first-strand cDNA synthesis, oligo(dT) primers are recommended because of their specificity for eukaryotic mRNA, and they allow many different targets to be studied from the same cDNA pool. Typically, oligo(dT) primers are strings of 12–20 deoxythymidines. We offer oligo(dT) in different lengths and formats for flexibility in your reverse transcription experiments. For target mRNA containing strong transcriptional pauses, random primers are better suited because they anneal throughout the target molecules. They are also an ideal choice for nonpolyadenylated RNA, such as bacterial RNA. For two-step RT-PCR, a mixture of oligo(dT) and random primers is often used to achieve the benefits of both primer types. SuperScript IV VILO master mix is provided with an optimized ratio of primers. Reverse transcription primers Learn more at thermofisher.com/rtprimers Reverse transcription Poly(A) tail Oligo(dT) primer Random primers 22 Table of contents PCR Table of contents 23 The polymerase chain reaction (PCR) amplifies DNA, allowing its detection from very low sample inputs. The process involves 3 steps that are repeated over multiple cycles: 1) Denaturation: double-stranded DNA is heated to separate the strands 2) Annealing: primers, short DNA molecules, bind to specific regions of the DNA 3) Extension: DNA polymerase extends the primers Find technical and educational resources about PCR at thermofisher.com/pcreducation. PCR 24 Table of contents VeriFlex Block temperature zones Helpful tip Using the right PCR plastics for your application and instrument can improve the reliability of your PCR results. Go to thermofisher.com/findplastics to determine the right PCR plastics for you. Thermal cyclers, which automate the heating and cooling cycles required to amplify DNA, play a critical role in the success of PCR. The following are things to consider when selecting a thermal cycler. Precise temperature control Thermal cyclers with precise temperature control enable you to quickly and accurately determine optimal annealing temperatures. Several block technologies, including gradient and Applied Biosystems™ VeriFlex™ Blocks temperature control, are available. A VeriFlex Block employs a separate heating and cooling element in each temperature zone, allowing better control and precision of temperatures. Learn more about the technology at thermofisher.com/veriflextechnology. Reliability Thermal cyclers should be able to withstand repeated use, environmental stress, and shipping conditions. Component reliability can be tested using robotic assemblies for repeated testing of frequently used instrument components such as the heated lid, touchscreens, and temperature cycling modules. Applied Biosystems™ thermal cyclers adhere to stringent reliability criteria, which are reported at thermofisher.com/thermalcyclerreliability. Temperature accuracy Thermal cycler temperature accuracy is a key factor in the success or failure of a PCR reaction. It is particularly important during annealing temperature optimization, Thermal cyclers which requires both accuracy and consistency in the thermal cycler block. If the temperature set point of the instrument does not correspond to the actual temperature of the block, further temperature optimization could be required. Review a study of temperature accuracy in a number of models, available at thermofisher.com/thermalcycleraccuracy. Features A variety of Applied Biosystems thermal cyclers are available to fit your applications and budget. Certain features may be important to you, depending on your needs. If you perform PCR optimization frequently, you will likely benefit from an instrument with a VeriFlex Block. If you would like to run optimized assays on a new or different thermal cycler, you can save re-optimization time by using a simulation mode. If you want remote access to your instrument, you will appreciate the convenience of cloud-enabled thermal cyclers. They allow you to design and share protocols, schedule an instrument, start or stop a run, and check run status from anywhere, on any mobile device or desktop computer. Fleet control If you manage multiple thermal cyclers and users, you may benefit from a single interface for viewing all instruments at a glance and setting custom permissions by instrument, user, and method. Learn more at thermofisher.com/fleetcontrol. Learn more at thermofisher.com/thermalcyclers Applied Biosystems™ Thermal Cycler Fleet Control Software PCR Table of contents 25 Select the Applied Biosystems thermal cycler that’s right for you Cloud-enabled Cloud-enabled Looking for a CE-IVD–labeled thermal cycler? Learn more about VeritiPro™ Dx Thermal Cycler at thermofisher.com/veritiprodx PCR Questions for customers ProFlex™ PCR System Do you share the device with colleagues? Do you expect your throughput needs to change? Do you want to access your instrument remotely? VeritiPro™ Thermal Cycler Do you perform a lot of optimizations? Do you want to access your instrument remotely? Key benefits Ultimate flexibility and throughput Ultimate performance Max sample throughput 768 reactions (2 x 384-well block) 480,000 reactions (flat block) 384 reactions Max block ramp rate 6.0°C/sec 6.0°C/sec Temperature optimization 6-zone VeriFlex Block on 96-well system 2-zone VeriFlex Block on 3 x 32-well system 6-zone VeriFlex Block on 96-well system Compatible with Fleet Control Software Yes Yes 3D tour thermofisher.com/proflex3dtour thermofisher.com/veritipro3dtour 26 Table of contents Cloud-enabled Automation-ready Find out more at thermofisher.com/thermalcyclers PCR MiniAmp™ Thermal Cycler Do you want an instrument with just the features needed for routine PCR? Do you want to access your instrument remotely? Automated Thermal Cycler Do you want to place your instrument on a robotic platform now or in the future? Routine PCR, elevated Designed for easy robotic integration 96 reactions 384 reactions 3.5°C/sec 3.5°C/sec 3-zone VeriFlex Block on MiniAmp Plus model None Yes Yes thermofisher.com/atc3dtour Table of contents 27 Since PCR is a sensitive detection method, PCR plastics must be of high quality and free of contaminants and inhibitors to enable optimal performance. Regardless of the plastics format, proper fit and uniform heat transfer during thermal cycling are essential. Manufacturing quality control Applied Biosystems™ PCR and qPCR plastic consumables are manufactured in world-class facilities dedicated to the production of high-quality molecular biology–grade plastics. After manufacturing, all plastics undergo strict quality control. Integrity testing: Each well of every plate is visually inspected and tested for leaks. This thorough quality control screening verifies every well is intact to protect all reactions. Evaporation testing: Samples are run through PCR to test sealing performance. Well liquid volumes are analyzed post-PCR to verify seal integrity. This helps ensure that every production lot conforms to strict tolerances. Biological testing: All PCR plastics from Thermo Fisher Scientific are tested and guaranteed to be free of DNA, RNases, nuclease inhibitors, and PCR inhibitors. PCR certificates are available for convenience and documentation purposes, upon request. EnduraPlate microplate for high-throughput robotic applications PCR and qPCR plastics, seals, and accessories Polycarbonate (hard shell) frame Skirt Polypropylene wells Materials Applied Biosystems™ MicroAmp™ optical microplates are made of polypropylene for optimal transfer of thermal energy for efficient PCR. A select medical‑grade polypropylene is utilized for its exceptional biocompatibility and inert properties. Applied Biosystems™ MicroAmp™ EnduraPlate™ microplates are constructed with a polycarbonate frame. This rigid design prevents damage from robotic grippers and provides better tolerance of rapid heating and cooling, while retaining thin-walled polypropylene wells for efficient heat transfer to the reaction mixture. Thermo Scientific™ Sustain™ Series— reduce the environmental impact of your lab Explore PCR plastics from the Sustain Series. Manufactured under ISO 9001 guidelines within a Class 100,000 cleanroom from a biobased medical-grade virgin polypropylene that is chemically and molecularly identical to existing Thermo Scientific™ PCR plastics, these products are certified by the International Sustainability and Carbon Certification (ISCC+) system. This production approach is more sustainable and is equivalent to 1.96x* lower CO2 equivalent emissions per kg of biobased polypropylene resin compared to traditional fossil fuel– based polypropylene resin. thermofisher.com/sustainplastics Find out more at thermofisher.com/pcrplastics PCR * Compared to equivalent fossil fuel–derived polypropylene resin emissions, cradle to gate (kg CO2e). 28 Table of contents Small-scale experiments with a few samples Daily experiments Reliable and sustainable PCR plastics Complete-workflow experiments— ideal for automation Automation-compatible PCR plastics product Single tubes, strips, caps, adhesive film, and accessories MicroAmp™ optical microplates Sustain Series products MicroAmp™ EnduraPlate™ optical microplates MicroAmp™ EnduraPlate™ optical reaction plates Formats • Single tubes • Single tubes with caps • 8-strip tubes with caps • 12-strip caps • 32-well • 48-well Fast • 96-well* • 96-well Fast* • 384-well* • 96-well (multiple) • 384-well • Strip tubes • Strip tube caps • Strip tubes with attached caps • 96-well • 96-well Fast • 384-well • 96-well full-skirted • 96-well • 96-well Fast • 384-well DNA-, RNase-, PCR inhibitor–free Yes Yes Yes Yes Yes Colors available Clear, or mixed packs containing red, orange, blue, and green Clear Clear Single-color packs (red, blue, green, yellow, or clear) and 5-plate sampler (one of each color) Clear Barcode available No Yes (1 or 2 sides) No Yes (3 sides) Yes (3 sides) Automationcompatible No Yes (for those with * above) Yes Yes Yes Applied Biosystems PCR and qPCR plastics are validated and tested for reliability and optimal performance. They are “Engineer Approved” for use with all Applied Biosystems thermal cyclers and qPCR instruments, and are available in a variety of 32-, 48-, 96-, and 384-well plates; tube strips; single tubes; caps; and seals. The table below provides a detailed comparison of each product. Easily find the PCR and qPCR plastics compatible with your instrument using the online selection tool at thermofisher.com/findplastics. Did you know? Low-profile plastics, also referred to as “Fast” tubes or plates, are generally required for fast (0.1 mL) thermal blocks. Fast plastics utilize lower volumes (0.1 mL) than the standard (0.2 mL) tubes or plates. The low profile minimizes the air space above the reaction, helping reduce the effects of evaporation and enhancing thermal conductivity. Learn more about PCR and qPCR plastics at thermofisher.com/pcrplastics-education. PCR Table of contents 29 DNA polymerase is an essential component for PCR because of its key role in synthesizing new DNA strands. Because of the sensitive and specific nature of PCR, it is important to choose high-quality enzymes and reagents to produce optimal results. The following are things to consider when choosing PCR enzymes. Specificity Nonspecific amplification is one of the major hurdles in PCR, since it can drastically impact the yield and sensitivity of target amplification. One way to help reduce nonspecific amplification is through the use of a hot‑start DNA polymerase, which utilizes an antibody or chemical modification so that the polymerase becomes active only at the high temperature of the denaturation step. In addition to improving specificity, a hot-start DNA polymerase increases yield and allows convenient roomtemperature setup for high-throughput applications. Thermostability Since thermal cycling is a key feature of the conditions that enable the repetitive chain reaction of amplifying DNA, thermostability of the DNA polymerase to be used is also an important feature. Highly thermostable DNA polymerases are recommended for amplifying GC-rich or long templates that often require prolonged high-temperature reactions. PCR reagents Did you know? The residual bacterial DNA in recombinant PCR enzymes poses challenges in microbial genome analysis, such as accurately detecting bacterial strains by 16S rRNA gene sequences. To enable confidence and success in microbial PCR assays, choose PCR enzymes with controlled low levels of residual bacterial and human genomic DNA. Find out more at thermofisher.com/broad-range-pcr. Helpful tip Direct PCR is a way to help simplify PCR experiments, save time, and prevent sample loss in the workflow. Direct PCR allows you to amplify target sequences directly from the samples without the need to first isolate and purify the DNA. Find out more at thermofisher.com/direct-pcr. Find out more at thermofisher.com/pcrenzymes Fidelity The fidelity, or proofreading capability, of a DNA polymerase is based on its 3ʹ to 5ʹ exonuclease activity, which corrects misincorporated nucleotides. This function is critical in applications such as cloning, sequencing, and site-directed mutagenesis, for accurate replication of DNA sequences. Processivity A DNA polymerase’s processivity is defined as the number of nucleotides being incorporated in a single binding event. This property often reflects synthesis rate and speed, as well as affinity for its substrates. Therefore, highly processive DNA polymerases are beneficial to amplify challenging templates such as long, GC-rich, or inhibitor-containing DNA. Primer annealing temperature The primer annealing temperature of each DNA fragment to be amplified often needs optimization when designing a PCR protocol. To help simplify annealing and enable co-cycling of PCR assays, consider a DNA polymerase with a reaction buffer that allows a universal annealing temperature of 60°C for primers. PCR 30 Table of contents Inactive Activated Antibody-based hot-start DNA polymerase and its activation in PCR for enhanced specificity Helpful tip One of the most common PCR troubleshooting issues is the presence of unwanted bands, or nonspecific amplification. To reduce nonspecific amplification: • Use hot-start PCR • Optimize annealing temperature • Check primer design • Prevent DNA cross-contamination • Decrease template and/or primer concentration • Optimize Mg2+ concentration PCR Did you know? High-quality dNTPs are essential for successful PCR reactions. Thermo Fisher is one of the few primary manufacturers of nucleotides. Our high-quality nucleotides include dNTPs, NTPs, and modified nucleotides to support PCR and related applications. Building your own master mix? Learn more about our offerings and robust quality testing. Find out more at thermofisher.com/dNTPs. Table of contents 31 Choose the right PCR reagent for your research needs DNA polymerase Invitrogen™ Platinum™ SuperFi™ II DNA Polymerase Invitrogen™ Platinum™ II Taq Hot-Start DNA Polymerase Applied Biosystems™ AmpliTaq Gold™ 360 DNA Polymerase Invitrogen™ Platinum™ Direct PCR Universal Master Mix PCR type High-fidelity PCR Hot-start PCR Hot-start PCR Direct PCR Capabilities Highly accurate amplicon sequences, universal primer annealing, robust amplification of difficult targets Universal primer annealing, fast DNA synthesis, detection of low-abundance targets Chemical hot start Detection of target DNA without genomic DNA purification Technical specifications Fidelity compared to Taq polymerase >300x 1x 1x 1x Target length Up to 20 kb* Up to 5 kb Up to 5 kb Up to 8 kb Hot-start modification Antibody-mediated Antibody-mediated Chemical modification Antibody-mediated Speed 15–30 sec/kb 15 sec/kb 60 sec/kb 20 sec/kb Universal primer annealing Yes Yes No Yes Inhibitor tolerance Yes Yes No Yes Blunt or 3´-A end Blunt 3´-A 3´-A 3´-A Compatible with Applied Biosystems™ TaqMan™ probes No Yes Yes No Certified low level of bacterial gDNA Yes Yes Yes No Applications Cloning and subcloning • Site-directed mutagenesis • GC-rich amplification • • • • Template generation for sequencing • • • • High-throughput PCR • • • Long PCR (up to 20 kb) • Genotyping • • • • Amplification of samples with suboptimal purity • • • Colony PCR • • • • Multiplex PCR • • • • Fast PCR • • • * Amplification of up to 40 kb fragment sizes is possible, but may require additional optimization of reaction conditions and primer design. A comprehensive portfolio of PCR enzymes and master mixes is available with the high performance and consistency you need. Start with the selection guide below to find the best enzyme for common PCR applications. PCR Learn more at thermofisher.com/pcrenzymes Did you know? These products are free of OPE and NPE, making them safer for aquatic life. 32 Table of contents To help your research move faster, we have continually improved our PCR enzymes and reagents. For example, the latest Invitrogen™ Platinum™ DNA polymerases are designed with the following key innovative features. More robust and versatile Advanced enzymatic engineering and methodology provide DNA polymerases with fast cycling, high tolerance of PCR inhibitors, and efficient amplification of challenging DNA like GC-rich sequences. These features help you amplify DNA targets confidently with speed and simplicity. Find out more at thermofisher.com/platinumenzymes. Universal primer annealing The innovative Invitrogen™ Platinum™ PCR buffers enable universal primer annealing at 60°C. This design allows you to co-cycle different PCR assays (instead of running them sequentially), drastically reducing tedious optimization steps and saving time. Find out more at thermofisher.com/universalannealing. Direct gel loading The latest Invitrogen™ Platinum™ DNA polymerase master mixes are available in a green buffer format that allows direct gel loading and eliminates tedious steps of dye addition, helping to reduce pipetting errors. DNA migration is easily tracked with two dyes (blue and yellow) that are readily visible during electrophoresis (depicted in the lanes for 5 and 15 min in the figure to the right). Innovations for superior PCR PCR Table of contents 33 With a complete range of custom-synthesized oligonucleotide primers, probes, and genes, we recognize the need for quality, reliability, and convenience. Invitrogen™ custom DNA oligos are synthesized on state-of-the-art automated systems to help increase performance, speed, and capacity. Available in a range of synthesis scales, purification options, and modifications, oligos are analyzed by mass spectrometry to help ensure the quality of all end products. This means you will receive high-quality custom DNA oligos quickly and efficiently. Choose the right oligos and purification methods for your applications at thermofisher.com/oligos. Best practices for primer design Good primer design is essential for a successful PCR assay. For design tips, review the infographic below or go to thermofisher.com/primerdesign. Custom DNA oligos Avoid low-complexity sequences in the template and primer, and runs of the same nucleotide Sequence TCGCGATATATATATC ATCGGGGGGTACTAG Avoid self-dimers (complementarity of >3 bases Avoid secondary structures in primer design 5–17 bp 18–22 b p 23–40 bp AC GCAGCAT3´ G CGT5´ T Length GC content (%) Annealing temperature (Tm) <55°C 55–65°C >65°C 5´ 5´ 3´ 3´ <40% 40–60% >60% Find out more at thermofisher.com/oligos PCR 34 Table of contents Purification method Description Benefit Applications 25 nmol 50 nmol 200 nmol 1 μmol 10 μmol Desalt 25 nmol, 10–100 nt; 50 nmol, 5–100 nt Oligos are processed through a normal‑phase chromatography column, which removes salts but not truncated sequences A salt-free DNA solution, ready to use; suitable for many PCR and sequencing applications without further purification • Endpoint PCR • Isothermal sequencing • Fluorescent sequencing • Microarrays • Amplified fragment length polymorphism (AFLP) analysis • • • • • Cartridge 50 nmol–1 μmol, 7–55 nt Based on reverse-phase chromatography; removes truncated sequences from the completed synthesis Provides full-length sequences necessary for some applications • Antisense oligos (ASO) • First-strand cDNA synthesis for generation of libraries • Fluorescent sequencing • Gel shift assays • PCR using oligos with critical 5´ sequences (e.g., restriction endonuclease sites, RNA polymerase promoters) • Production of cloning adapters • Site-directed mutagenesis NA • • • NA HPLC ≥50 nmol, 10–55 nt; long oligo HPLC available Reverse-phase high-performance liquid chromatography (HPLC) removes truncated sequences or unincorporated labels the same way as cartridge purification Ensures highly purified primer required in some applications (≥85% full length) NA • • • • PAGE ≥200 nmol, 7–100 nt Polyacrylamide gel electrophoresis (PAGE) is a method used to differentiate fulllength product from truncated sequences based on size and conformation Provides the highest percentage of full-length oligos (≥85%) required for certain demanding applications such as mutagenesis or adapter production NA NA • • • * Pricing and “Add to cart” feature may vary by geographic region. For questions, please contact your local office or distributor at thermofisher.com/contactus. Primer design made easy Whether you’re performing PCR, cloning, or capillary electrophoresis (CE) sequencing, take advantage of the benefits offered by our robust and easy-to-use Primer3-based Invitrogen™ OligoPerfect™ Designer. • Speed up—design primers for up to 50 genes at the same time • Store your data—ability to save your projects • Work smarter—recognizes .txt and .fasta file types • Order with ease—select and add primers directly to your online cart from the design tool* Try the OligoPerfect Designer at thermofisher.com/oligoperfect or visit the oligo utility hub for our full suite of tools and calculators at thermofisher.com/oligotools. PCR Did you know? Thermo Fisher Scientific offers scales beyond 10 μmol up to kilograms, and employs a team of manufacturing scientists dedicated to custom method development for unique modifications. For information on large-scale and complex project capabilities, visit thermofisher.com/largescaleoligos. Table of contents 35 Electrophoresis 36 Table of contents Find technical resources on nucleic acid electrophoresis at thermofisher.com/na-electrophoresis-education Nucleic acid electrophoresis is a common technique in molecular biology used to separate, identify, quantify, and purify nucleic acids. Setting up electrophoresis involves a number of steps to achieve optimal separation and analysis of nucleic acid samples, such as gel preparation, ladder selection, sample visualization, and gel documentation. Table of contents 37 If you need … Convenience, rapid results, and a safer workflow High-quality reagents, a versatile workflow, and cost savings Product E-Gel Agarose Gels UltraPure Agarose Product format Precast agarose cassettes Powder Buffer Dry—none* TBE or TAE Protocol time (approx.) 18 min 120 min Ready to use Yes No Get more information at thermofisher.com/egel thermofisher.com/ultrapure * Note: This is a dry precast electrophoresis system. Choosing the right tools for nucleic acid electrophoresis can significantly improve and accelerate results, enabling you to address downstream applications sooner. Determining the appropriate gel type and gel concentration is an essential step that will help streamline the separation of nucleic acids. Learn more about convenient reagents for agarose gel electrophoresis, including hassle-free precast Invitrogen™ E-Gel™ Agarose Gels and pour-your-own Invitrogen™ UltraPure™ Agarose reagents, in this section. Nucleic acid electrophoresis ~18 minutes Prepare gel solution (weigh, pour buer, melt) (10 min) Assemble tray, pour and set gel (add stain, pour gel, add comb, cool) (45 min) Load samples (10 min) Run gel (50 min) Image and document (5 min) ~120 minutes Prepare E-Gel precast agarose electrophoresis system (3 min) Run E-Gel EX cassette (8 min) Load samples (5 min) Image and document (2 min) ~18 minutes Prepare gel solution (weigh, pour buer, melt) (10 min) Assemble tray, pour and set gel (add stain, pour gel, add comb, cool) (45 min) Load samples (10 min) Run gel (50 min) Image and document (5 min) ~120 minutes Prepare E-Gel precast agarose electrophoresis system (3 min) Run E-Gel EX cassette (8 min) Load samples (5 min) Image and document (2 min) Find out more at thermofisher.com/electrophoresis Electrophoresis Traditional DNA electrophoresis workflow E-Gel precast agarose electrophoresis system 38 Table of contents Restriction digest analysis: • Fast and easy preparation of vectors for cloning experiments using FastDigest restriction enzymes and the Invitrogen™ E-Gel™ Power Snap Plus Electrophoresis System RNA analysis: • Optimization of loading conditions of RNA markers on an Invitrogen™ E-Gel™ system Genotyping: • Convenient and accurate visualization of DNA fragments, such as universal target fragments, for genotyping E-Gel precast gels Using precast agarose gels can simplify the nucleic acid electrophoresis workflow. Invitrogen™ E-Gel™ precast gels are self-contained and ready for use with the agarose, electrodes, and DNA stain packaged inside a disposable cassette. There are no gels to pour, buffers to make, staining or destaining steps to perform, or gel boxes to assemble. Just load your samples and run. E-Gel precast gels offer excellent resolution and clarity in ≤18 minutes and are ideal for analyzing PCR products, restriction digests, plasmid preparations, and genotyping products. To help simplify cloning workflows, Invitrogen™ E-Gel™ CloneWell™ II gels use a double-comb design to enable recovery of purified DNA for downstream applications, without the need for additional purification kits or steps. Find out more at thermofisher.com/egel. Simplify electrophoresis with E-Gel precast agarose cassettes E-Gel precast gels—faster and safer workflows Helpful tips E-Gel precast gels are available in a variety of formats for routine and high-throughput applications, with different stains and agarose percentages (0.8%, 1%, 2%, and 4%). To find the right gel for your needs, see the selection guide at thermofisher.com/egelselection. Choose Invitrogen™ E-Gel™ DNA ladders for precise electrophoresis band analysis with exceptional DNA fragment purity and quality, reduced dye masking, and improved ladder migration on E-Gel precast agarose gels. Find out more at thermofisher.com/egel-ladders. Find out more at thermofisher.com/egel Electrophoresis Table of contents 39 Fast and accurate analysis of RNA with E-Gel EX agarose gels As RNA is the starting material for many workflows, its quality is important. Because RNA tends to form secondary structures and is prone to degradation, traditional methods of agarose gel electrophoresis are challenging. Traditional methods require additional time for buffer preparation, hand-pouring of gels, and prolonged run times. Because denaturing agents need to be added, gels are run in a fume hood. With Invitrogen™ E-Gel™ EX Agarose Gels, several labor-intensive steps are removed from this process. The use of toxic denaturing reagents in the gel is no longer required. To denature the RNA, samples are treated with 50% formamide and heated at 70°C for 10 min, and then chilled on ice prior to loading on the E-Gel EX gel. The figure to the right shows clear, crisp bands from a diverse collection of RNA samples. Lanes 1–8 are samples, lanes M and 9 are ladders. Genotyping detection of GMOs in soy products Invitrogen™ E-Gel™ 48 and 96 Agarose Gels are useful for the rapid testing of larger numbers of samples. In this example, store-bought foods with unknown GMO status were tested. Multiplex PCR was used to amplify a known GMO sequence and an internal control. A sample with a known genetic modification was used as a positive control (F1). The PCR products were separated and visualized on an E-Gel 48-well 2% agarose gel with Invitrogen™ SYBR™ Safe stain. Read the application note for more details. Two bands indicate the presence of genetic modification in many of the samples. E-Gel application examples Electrophoresis Helpful tips Improve your nucleic acid electrophoresis. View our webinar Nucleic Acid Agarose Gel Electrophoresis: Techniques, Innovations, and Common Errors 40 Table of contents Select your E-Gel electrophoresis device Cloud-enabled Ethernet connectivity Integrate your electrophoresis running and imaging into a single small device Enjoy speed and convenience Which system is right for you? Invitrogen™ E-Gel™ Power Snap Plus Electrophoresis System Do you use electrophoresis often? Would you like to store your gel images on internal servers or the cloud? Do you want to perform quantitative analysis of your gels? Do you value speed? Would you like to store, share, and analyze gel images online? Invitrogen™ E-Gel™ Power Snap Electrophoresis System Do you run less than 25 samples at a time? Do you want a simple and fast solution for your electrophoresis? Do you need to save bench space? Key difference Low- to high-throughput analysis Low-throughput analysis Memory 64 GB 32 GB Connectivity USB drive, ethernet, Wi-Fi, printer USB drive Sample throughput Up to 96 samples Up to 22 samples Applications Genotyping, fast PCR analysis, routine electrophoresis, and cloning Routine electrophoresis and cloning Software Invitrogen™ iBright™ Analysis Software NA Find out more thermofisher.com/powersnapplus thermofisher.com/powersnap Electrophoresis Table of contents 41 Fluorescent nucleic acid gel stains Safer detection Ultimate detection SYBR Safe stain SYBR Gold stain Sensitivity (dsDNA) Sensitive (>3 ng) Ultrasensitive (>0.1 ng) Less hazardous and more environmentally friendly • Improved cloning efficiency • • Electrophoresis reagents Choose high-quality agarose to pour your own gels. Select a ladder of the correct size range and an improved DNA stain for best detection and size estimation of DNA fragments. DNA stains Detection of nucleic acid samples in gels can be improved using fluorescent dyes that are safer or more sensitive than ethidium bromide. Invitrogen™ SYBR™ Safe and SYBR™ Gold stains provide greater safety or sensitivity with lower background fluorescence than the conventional ethidium bromide stain. SYBR Safe stain is specifically formulated to be less hazardous than ethidium bromide and reduces your exposure to UV light. Find out more at thermofisher.com/stains and thermofisher.com/sybrsafe. Did you know? Chromatographically purified nucleic acid fragments are considered the trusted standard for ladders since the technology provides high control over quality, banding pattern, intensity, and quantity for ladder composition. Learn more at thermofisher.com/na-electrophoresis-education. UltraPure reagents for electrophoresis Invitrogen™ UltraPure™ reagents are specifically formulated to help meet your nucleic acid analysis and purification needs. Invitrogen™ UltraPure™ Agarose and other reagents are made from highly pure biochemicals for maximum reliability and superior performance. Find out more at thermofisher.com/ultrapure. DNA ladders Invitrogen™ DNA ladders are available in a wide variety of size ranges (10 to 48,502 bp) and formats for different applications. To create DNA ladders of exceptional quality, each fragment is purified individually using proprietary chromatography-based technology. Our DNA ladders are stable during prolonged storage at room temperature and after multiple freeze-thaw cycles. Find out more at thermofisher.com/ladders. Electrophoresis 42 Table of contents Cloning Table of contents 43 Molecular cloning and synthetic DNA assembly are essential techniques in biotechnology for DNA sequence assembly. Restriction enzymes, TOPO PCR cloning, Gibson Assembly cloning, Golden Gate cloning, and Gateway cloning enable precise assembly of simple and complex constructs. Find technical resources on molecular cloning at thermofisher.com/cloningeducation 44 Table of contents From restriction enzymes to gene synthesis, a large portfolio of tools and resources is available to help you obtain high-quality cloned DNA for your next discovery. Method Thermo Scientific™ FastDigest™ restriction enzymes Invitrogen™ TOPO™ cloning Invitrogen™ Gateway™ cloning Invitrogen™ GeneArt™ seamless cloning and GeneArt™ Gibson Assembly™ cloning kits Invitrogen™ GeneArt™ Type IIS assembly Invitrogen™ GeneArt™ Strings™ DNA Fragments Invitrogen™ GeneArt™ Gene Synthesis Key benefits • Familiarity, flexibility, convenience, time savings • Universal protocol and complete digestion in 5–15 minutes in one buffer • 100% buffer compatibility with downstream applications • Direct loading on gels • >95% efficiency, 5-minute PCR cloning • Available in a variety of formats and sizes • High-throughput and high-efficiency shuttling among multiple expression vectors • Seamless multifragment assembly by homologous recombination • Directional cloning of up to 15 fragments • Up to 95% efficiency and 15-minute cloning • One-tube seamless multifragment assembly by simultaneous restriction digestion and ligation • Directional cloning of up to 8 fragments, for up to 20 kb total • Efficient for repetitive and very small sequences • Synthesized DNA fragments ready to clone via the method of your choice • No starting DNA required • Pool sequence–verified • Custom-cloned genes in your choice of vector • Sequence-verified • Can be optimized for a specific host for maximal protein expression • No starting DNA required Technology basics • Restriction digestion and ligation • Topoisomerase-based, ligase-free cloning • Single-step, directional, and site-specific DNA recombination • Restriction enzyme– and ligase-free • End-terminal homology recombination using overlapping sequences • Transformationassociated recombination (TAR) in Saccharomyces cerevisiae • Type IIS restriction and ligation in a single reaction • Linear dsDNA assembled from pooled synthetic oligonucleotides • 200–3,000 bp, also available in library format with randomized bases • DNA of interest cloned in vector • 100% sequence-verified with quality assurance documentation Online tools available FastDigest selection tool TOPO PCR selection tool Vector list Invitrogen™ GeneArt™ s Primer and Construct Design Tool Invitrogen™ GeneArt™ s Instant Designer for design and optimization Needs DNA source material (gene in plasmid, library, etc.) • • • • • Use your own vector • * • • • • * Vector needs to be converted with Invitrogen™ Gateway™ Vector Conversion System with One Shot™ ccdB Survival™ 2 T1R Competent Cells. Cloning and gene synthesis Cloning Discover more at thermofisher.com/cloning Sequenced, cloned genes Assemble or clone multiple fragments Create large inserts from multiple fragments Express genes in multiple systems Clone PCR fragments Move fragments from one vector to another Table of contents 45 Restriction enzyme cloning Comparison of digestion efficiencies of restriction enzymes. FastDigest restriction enzymes (A) digest plasmid DNA much more efficiently than supplier N’s enzymes (B). In this experiment, 1 μg each of plasmid DNA was digested for ~15 min with the indicated restriction enzymes, using supplier N’s protocol. Incomplete digestion is shown in red boxes. 1: Undigested plasmid DNA 2: SpeI-HF 3: XbaI 4: NdeI 5: SalI-HF 6: AvrII 7: BsaI 8: EaqI-HF 9: BglI 1 2 3 4 5 6 7 8 9 Plasmid DNA digested with FastDigest enzymes 1: Undigested plasmid DNA 2: FastDigest BcuI 3: FastDigest XbaI 4: FastDigest NdeI 5: FastDigest SalI 6: FastDigest XmaJI 7: FastDigest Eco31I 8: FastDigest Eco52I 9: FastDigest BglII 1 2 3 4 5 6 7 8 9 Plasmid DNA digested with supplier N’s enzymes Found naturally in bacteria, restriction enzymes recognize and cleave specific DNA sequences. Cleavage results in “sticky” ends (5ʹ or 3ʹ protruding ends) or blunt ends that allow DNA inserts to be cloned into vectors with compatible ends. Star activity, buffer compatibility, and varying protocols for complete digestion are some common hurdles in restriction digestion. FastDigest restriction enzymes To simplify cloning, we offer FastDigest enzymes—an advanced line of restriction enzymes that share buffer compatibility with downstream modifying enzymes. Benefits include: • Complete digestion in 5–15 min • Universal buffer for multiple digestions for any combination of enzymes • No sequential digestions and buffer changes • 176 unique enzymes • Direct loading of reaction mixture on gels See our enzyme selection tool at thermofisher.com/fastdigest. DNA/RNA-modifying enzymes Complete your cloning reaction with modifying enzymes that have 100% activity in FastDigest and FastDigest Green buffers. These include: • Thermo Scientific™ T4 DNA Ligase—used to join the ends of DNA fragments and vectors in a fast 10 min reaction • Thermo Scientific™ FastAP™ Thermosensitive Alkaline Phosphatase—used for the dephosphorylation of cloning vectors to prevent recircularization. Fast 10 min reaction with complete inactivation in a 5 min reaction • Thermo Scientific™ T4 Polynucleotide Kinase—phosphorylates PCR products, oligonucleotides, and other DNA prior to ligation reactions • Thermo Scientific™ DNA polymerases—used for DNA blunting by filling in 5ʹ-overhangs and second-strand synthesis of cDNA • Invitrogen™ CorrectASE™ enzyme—utilized to enhance the accuracy of synthetic gene and DNA fragment assembly by removing mismatches A B Cloning Restriction enzyme Sticky ends Doublestranded DNA Visit thermofisher.com/modifyingenzymes. 46 Table of contents Type IIS restriction enzymes A specific group of restriction enzymes called Type IIS endonucleases cleave DNA outside of their recognition sequences. In combination with DNA ligase, Type IIS restriction enzymes are utilized to drive the insertion of one or several DNA fragments into a recipient vector without the inclusion of residual restriction enzyme sites and other unwanted DNA sequences at fragment junctions (scarless cloning). Commonly referred to as Golden Gate cloning, this method is useful for the construction of large DNA fragments. Find FastDigest Type IIS enzymes at thermofisher.com/fastdigesttypeiis. For GeneArt Type IIS Assembly Kits, go to thermofisher.com/typeiis. Cloning Did you know? Both Gateway and Type IIS restriction enzyme (Golden Gate) cloning technologies have been used for CRISPR-based genome editing in plants. View an article and references here. Table of contents 47 Did you know? The Invitrogen™ TOPO™ XL-2 Complete PCR Cloning Kit provides all the necessary elements for highly efficient cloning of extra-long PCR products from 1 to 13 kb. thermofisher.com/topoxl2 PCR cloning Perform PCR with Taq or proofreading polymerase Add 1 μL of PCR reaction to 1 μL of TOPO cloning vector TOPO cloning vector Incubate 5 minutes at room temperature Perform transformation with provided competent E. coli strain TOPO TOPO PCR product PCR product A CACC A PCR product PCR cloning is a method in which double-stranded DNA fragments amplified by PCR are ligated into a vector. With PCR amplification, this cloning technique requires much less starting material for the insert sequence and allows introduction of new restriction and/or recombination sites to the 5ʹ end of the inserts. TOPO PCR cloning requires just three easy steps. Entry clone attL1 attL2 KanR Expression clone attB1 attB2 AmpR + + Destination vector attR1 attR2 AmpR ccdB Donor vector attP1 attP2 KanR ccdB BP Clonase LR Clonase Gateway cloning To shuttle a DNA fragment of interest (insert) among vectors, the Gateway cloning system offers site-specific, recombinase-based cloning. It maintains the insert’s proper orientation and reading frame during shuttling using the Gateway vectors. Once a gene is cloned into an entry clone, you can then move the DNA fragment into one or more destination vectors simultaneously. Find out more at thermofisher.com/gatewaycloning. TOPO cloning TOPO PCR cloning technology was developed to help improve cloning efficiency, simplify protocol setup, and accommodate a wide range of PCR insert sizes. TOPO cloning vectors are linearized by the activity of topoisomerase I (which also has a ligase function) that is covalently bound to the 3ʹ phosphate on each end (see figure below). This system enables the vectors to be joined to PCR inserts with compatible ends (with up to 95% efficiency), without the need for additional ligation steps, in 5 minutes. Find out more at thermofisher.com/topo. Quickly find your TOPO cloning kit with our interactive selection tool. Search by application, vector, or desired competent cells at thermofisher.com/topoguide. Cloning Gateway cloning system reactions. The scheme shows the four types of plasmids and enzyme mixes involved in Gateway cloning reactions. Red arrow represents the fragment of interest. Adapted from Katzen F (2007) Expert Opin Drug Discov 2(4):571–589. 48 Table of contents GeneArt Gibson Assembly and seamless cloning kits Several methods are available to assemble multiple PCR or synthesized DNA fragments. These include seamless cloning, Type IIS restriction enzyme (Golden Gate) cloning, and Gibson Assembly cloning. Using our kits, cloning can typically take place within half a day. Invitrogen™ GeneArt™ Gibson Assembly™ kits allow for the simultaneous assembly of up to 15 very large DNA fragments (up to a total of 100 kb) to create precise and seamless constructs with no additional sequences, in highly efficient reactions. This cloning method circumvents the need for multiple rounds of restriction enzyme analysis and digestion, DNA end repair, dephosphorylation, ligation, enzyme inactivation, and cleanup, and is a powerful tool in synthetic biology. GeneArt Gibson Assembly kits offer these benefits: • Assembly of up to 15 fragments to build seamless clones • Cloning efficiencies up to >95% • Choice of complete kits with competent cells or master mixes Invitrogen™ GeneArt™ Seamless Cloning and Assembly Kits allow the assembly of up to 4 DNA fragments. The Invitrogen™ GeneArt™ Seamless PLUS Cloning and Assembly Kit comes with two linearized vectors for expression in either E. coli or Saccharomyces cerevisiae. DNA fragments Assemble DNA fragments and linear vector Assembled circular construct One Shot TOP10 chemically competent or ElectroMAX DH10B electrocompetent E. coli transformation mix Linear vector Did you know? The Gibson Assembly method has been referenced in thousands of peer‑reviewed publications and is a powerful method that can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes [1]. 1. Gibson DG et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. Discover more at thermofisher.com/seamless Cloning Comparison of seamless cloning kits Invitrogen product GeneArt™ Seamless Cloning and Assembly Kit GeneArt™ Seamless PLUS Cloning and Assembly Kit GeneArt™ Type IIS assembly kits GeneArt™ Gibson Assembly™ S HiFi and EX Kits Number of fragments for simultaneous assembly Up to 4 Up to 4 Up to 8 Up to 6 (HiFi) and 15 (EX) Reaction time 30 min 15–60 min 5–60 min 15–60 min Homologous overlaps 15 nt 15–80 nt 4–6 nt >20 nt Ligase in the master mix No No Yes Yes Vector included Yes (pUC19L) Yes (pYES7L and pUC19L) Yes (pType IIS recipient vector) No Mutagenesis protocol Yes Yes No Yes Table of contents 49 Save time by ordering your gene cloned into a vector of interest. Your gene can also be delivered as a DNA fragment similar to a PCR product. The only starting material required is the sequence. GeneArt gene synthesis and cloned genes Using the Invitrogen™ GeneArt™ Custom Gene Synthesis service is a reliable and cost-effective way to obtain customized DNA constructs with 100% sequence accuracy. The GeneArt gene synthesis service offers a variety of options, including accelerated production, cloning into your vector of choice, and plasmid preparation. • An industry leader for reliable delivery and performance • Proprietary, peer-reviewed, and widely used Invitrogen™ GeneArt™ GeneOptimizer™ algorithm optimizes genes for maximum protein expression, at no additional cost • High-quality production with ISO 9001:2015 certification • Delivery in tube format with a minimum of 5 μg DNA GeneArt Strings DNA fragments Invitrogen™ GeneArt™ Strings™ DNA fragments are an economical and time-saving alternative to PCR. They are made by the same high-quality process used for gene synthesis, and they can be prepared quickly for cloning. • Up to 3 kb available with fast production • Can be optimized with the GeneArt GeneOptimizer algorithm • Delivered dried, ready for resuspension and cloning with any method Cloning with synthetic DNA Oligonucleotide synthesis Bioinformatics Cloning Sequencing Final documentation Gene assembly GeneArt Gene Synthesis GeneArt Strings DNA Fragments 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 5 Ready for shipment Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 5 Ready for shipment Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 5 Ready for shipment Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 5 Ready for shipment Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight 555555555555555 G A A G G A G www A C G C A Day 3 Cloning Day 2 GeneAssembler process Day 4 Sequencing and quality control Day 5 Ready for shipment Day 1 Ordering until 3:00 p.m. (CET) Oligo synthesis overnight Find out more at thermofisher.com/genesynthesis Cloning 50 Table of contents Once the DNA fragment is cloned into a vector, transformation into bacteria is performed to enable propagation of sufficient quantities of the cloned DNA for downstream experiments. Selection of competent cells for transformation depends upon the transformation methods, strain genotypes, plasmid characteristics, and desired applications. Visit thermofisher.com/compcells-education for technical resources on competent cells. Choosing Invitrogen™ competent cells based on the application Transformation Is this for preparing singlestranded DNA (ssDNA)? Yes Yes Yes No No Is this for routine cloning and subcloning? Routine cloning Is high transformation eciency needed? * Stbl4 and OmniMAX T1R competent cells also contain the F´ episome. Note: Available in various packaging formats to meet throughput needs (bulk, One Shot™, MultiShot™ competent cells). More information can be found here. Is this for gDNA or library production? Is this for expression of nontoxic genes? Is this for unstable inserts? Is this for methylationsensitive plasmid DNA? Is this for Invitrogen™ Gateway™ destination vector propagation? Is this for expression of toxic proteins? Is this for improved regulation of protein expression? Note: Use GeneOptimizer™ tool to optimize sequence for higher protein expression in E. coli without losing protein function. For more information, contact technical support Is this for use with plasmids containing the R6K ori? Is this for maintaining supF-containing vectors? Yes Yes Yes Yes Yes No No No No Subcloning OmniMAX™ 2 T1R TOP10 Electrocomp™ E. coli ElectroMAX™ DH10B™ ElectroMAX™ DH10B™ T1R MegaX DH10B™ T1R ElectroMAX™ DH5„™-E gDNA or library production TOP10 ™ DH10B™ ™ DH10B™ T1R ™ DH5„™ ™ DH5„™ T1R Mach1™-T1R ™ Stbl2™ One Shot™ Stbl3™ ElectroMAX™ Stbl4™ Unstable inserts and lentiviral vectors Methylation-sensitive INV110 Gateway destination vector Expresses the pir gene T7 promoter T7 inhibitor Arabinose-inducible promoter Carries P3 plasmid ccdB Survival™ 2 T1R PIR1 PIR2 BL21 (DE3) BL21 Star™ (DE3) BL21 (DE3) pLysS BL21 (DE3) pLysE BL21 Star™ (DE3) pLysS BL21-AI™ TOP10/P3 MC1061/P3 Carries F´ episome* TOP10F´ Electrocomp™ ElectroMAX™ DH12S ™ DH5„F´IQ INV„F´ Protein production Yes No Yes No No Specialized cloning DH5„™ Cloning No No Yes Yes No For more information, contact technical support Electrocompetent cells Find out more at thermofisher.com/compcells Cloning Table of contents 51 Medium- and high-throughput transformation Performing bacterial transformations one by one can be very time-consuming and create a bottleneck in your experimental workflow. There are times when medium- and high-throughput transformation options are desired. Invitrogen™ MultiShot™ chemically competent cells provide three flexible product formats to meet your throughput needs. Find out more at thermofisher.com/multishot Bacterial growth media Sterile, quality growth media and plates are essential for selection and growth of transformed cells. Premade solutions offer convenience and consistency for your cloning workflows. Featured products include: • Invitrogen™ One Shot™ LB Agar Plates with or without Antibiotics—ready-to-use pre-poured plates with a longer shelf life of 3.5 months • Gibco™ LB Broth (1X) and Gibco™ Terrific Broth—popular growth media, available as convenient ready-to-use liquids • Invitrogen™ MagicMedia™ E. coli Expression Medium—autoinduction medium for protein expression in E. coli Other available media include Invitrogen™ S.O.C., 2-YT Broth, M9 Minimal Salts, and Gibco™ Bacto™ CD Supreme Fermentation Production Medium (FPM). Visit thermofisher.com/growthmedia to learn more. FlexPlate format • High-throughput option • 96-well plate separates into 12 x 8-well segments • Manual and automated platform transformations • Six E. coli strains available StripWell format • Medium-throughput option • Twelve 8-tube strips • Suitable for 1–96 transformations • Five E. coli strains available Did you know? Invitrogen competent cells can be provided in custom configurations per your request. Large and custom volumes as well as multiple formats are at your fingertips. Simply email us at customorders@thermofisher.com. Did you know? One Shot LB Agar Plates are available with a 3.5-month shelf life. Learn more at thermofisher.com/growthmedia. Transformation (cont.) Cloning 96-well plate • Highest-throughput option • Five 96-well plates • Available with the TOP10 strain • Stable replication of high copy number plasmids One Shot LB Agar Plates 52 Table of contents Isothermal amplification Table of contents 53 Isothermal amplification is a technique that utilizes enzymes, typically strand-displacing polymerases, to amplify nucleic acid sequences at a constant temperature. This results in continuous and exponential amplification that is not limited by the constraint of thermal cycling. Find technical and educational resources about isothermal amplification at thermofisher.com/isothermal 54 Table of contents Isothermal nucleic acid amplification techniques (INAATs) are fast alternatives to PCR that enable exponential amplification of nucleic acids at constant temperatures. PCR requires repeated denaturation steps at 95°C to separate the DNA strands for the primers to bind. In contrast, INAAT uses DNA polymerases with strand-displacing properties. This eliminates the need for temperature cycling, resulting in reaction times that are as short as 15 min. There are many known INAATs, including loop-mediated amplification (LAMP), multiple displacement amplification (MDA), whole genome amplification (WGA), recombinase polymerase amplification (RPA), and more. Different methods use specific enzymes and reaction conditions. Isothermal amplification overview Isothermal amplification Comparison of seamless cloning kits Technology Reaction temperature Reaction time Detection method Loop-mediated isothermal amplification (LAMP) 60–65°C 15–60 min Fluorescence, colorimetry, turbidity, lateral flow Multiple displacement amplification (MDA) 30–40°C 60–180 min Fluorescence, colorimetry Whole genome amplification (WGA) 30–40°C 60–180 min Fluorescence, colorimetry Recombinase polymerase amplification (RPA) 37°C 30–60 min Fluorescence, lateral flow Rolling circle amplification (RCA) 30–65°C 60–90 min Fluorescence, colorimetry, turbidity Once assembled, isothermal reactions can be run on a heat block, thermal cycler, or real-time PCR instrument. Using a real-time PCR instrument also enables fluorescent detection of reaction products. Alternatively, positive results can be detected visually using DNA-binding dyes like Invitrogen™ SYBR™ Green Nucleic Acid Gel Stain. Many instruments, like microplate readers, can be also used to measure the dye-labeled reaction products. Reactions can be further analyzed by agarose gel electrophoresis. Table of contents 55 LAMP overview One commonly used isothermal amplification technique is loop-mediated isothermal amplification (LAMP), which utilizes a set of four to six primers and a strand-displacing polymerase, such as Invitrogen™ Lyo-ready Bst DNA Polymerase, to amplify target DNA at a constant temperature. The LAMP reaction produces a large amount of DNA amplicons, with a characteristic ladder-like pattern that can be visualized by gel electrophoresis, or that can be detected by turbidity, fluorescence, or colorimetry. SuperScript IV RT-LAMP Master Mix Invitrogen™ SuperScript™ IV RT-LAMP Master Mix is a reverse transcription loop‑mediated isothermal amplification (RT-LAMP)-based solution for fast and simple detection of various pathogens, including influenza virus, measles virus, S. enterica, S. aureus, SARS-CoV-2, and others. Our master mix reagents provide maximum flexibility to help optimize and accelerate your pathogen research and surveillance. Product highlights • Fast—RNA and DNA target detection in as little as 5 minutes with evolved Bst DNA polymerase • Efficient—one-step reaction for reverse transcription of RNA to cDNA with Invitrogen™ SuperScript™ IV Reverse Transcriptase • Sensitive—greater sensitivity and specificity utilizing Invitrogen™ RNaseOUT™ Recombinant Ribonuclease Inhibitor and an optimized buffer • Simple—streamlined workflow: single-tube format, only requires a 65°C heating block • Flexible—several options for evaluating results, including real-time and endpoint detection methods Loop-mediated isothermal amplification (LAMP) 3´ 5´ 3´ 3´ 3´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ Loop-mediated isothermal amplification (LAMP) Template F3 F3c F2c F2 F2 F2 F2 F2c F1c F1c F1 F1c F1c F1c F1 F1 F1c B1 B2 B2 B2 B2 B1 B1 B3 B1c B1c B1c B1c B1c B2c B2c B3c FIP B3 Loop F Loop B BIP and The amplification process begins with the invasion of an inner primer into the target nucleic acid sequence, followed by extension via a strand-displacing DNA polymerase. As the extension proceeds, the first product is displaced, and an outer primer anneals to the newly synthesized strand, forming a self-hybridizing loop structure. This structure contains multiple sites for amplification initiation and serves as a seed for exponential LAMP reactions. Find out more at thermofisher.com/lamp. Find out more about SuperScript IV RT-LAMP Master Mix here. Isothermal amplification 56 Table of contents Lyo-ready Bst DNA Polymerase Lyo-ready Bst DNA Polymerase is an engineered version of the Bst DNA polymerase large fragment, which shows a significantly faster reaction speed, increased sensitivity, and tolerance to inhibitors. Lyo-ready Bst DNA Polymerase provides maximum flexibility to optimize your LAMP reaction and works with various types of pathogens, including human adenovirus, measles virus, SARS-CoV-2, and other pathogens. Product highlights • Fast—amplifies targets in as little as 10 minutes • Sensitive—provides sensitivity down to 50 copies • Robust—amplifies even from inhibitor-containing RNA/DNA samples • Flexible—provides the ability to optimize your LAMP or RT-LAMP reaction Find out more about Lyo-ready Bst DNA Polymerase here Tips: 1. Did you know? The term “lyo-ready” refers to an enzyme that is provided in a liquid formulation without glycerol, making it compatible with microfluidics-based systems and various downstream applications such as lyophilization. Furthermore, it maintains the necessary stability and activity levels for direct enzymatic reactions. 2. To minimize nonspecific amplification in LAMP, follow these steps: a. Prevent cross-contamination: Use uncontaminated reagents and maintain a clean work environment. b. Enhance primer design: Optimize primer sequences for improved specificity. c. Optimize reaction conditions: Adjust Lyo-Ready Bst DNA Polymerase amount and reaction time. Isothermal amplification Table of contents 57 WGA is a popular technique used to amplify small amounts of DNA, as little as that of a single cell, to obtain large quantities of product for downstream applications like next-generation sequencing. This method is based on multiple displacement amplification (MDA). When the starting material is circular, RCA is utilized. Both MDA-WGA and RCA use a strand-displacing DNA polymerase. Thermo Fisher Scientific offers two types: the wild-type Thermo Scientific™ phi29 DNA Polymerase, and the engineered Thermo Scientific™ EquiPhi29™ DNA Polymerase, which has exceptional performance compared to phi29 DNA Polymerase. 3´ 3´ 3´ 3´ 3´ 3´ 5´ 5´ 5´ 5´ 5´ 5´ 5´ 5´ 3´ 5´ 5´ 5´ 3´ 5´ 5´ Random primers Whole genome amplification (WGA) Polymerization DNA Strand-displacing DNA polmerase Strand displacement Polymerization and strand displacement Continued exponential amplification 5´ 5´ 3´ Multiple displacement amplification–whole genome amplification (MDA-WGA) The process of MDA-WGA starts with the addition of random hexamer primers to the DNA sample, which hybridize to the template and initiate DNA synthesis. A strand-displacing polymerase then synthesizes DNA strands along the template from the multiple priming positions. Isothermal amplification Rolling circle amplification (RCA) and whole genome amplification (WGA) Learn more about rolling circle amplification at thermofisher.com/rca 58 Table of contents Primer Strand-displacing polymerase Padlock probe Hybridization Ligation Circular template Linear RCA Rolling circle amplification (RCA) Ligase Target Rolling circle amplification (RCA) The process of RCA uses a circular DNA template, such as a plasmid or circularized oligonucleotide. DNA polymerase continuously synthesizes new DNA strands as it moves around the template. This leads to exponential amplification and the generation of concatemers containing numerous tandem repeats that are complementary to the circular template. Isothermal amplification EquiPhi29 DNA Polymerase EquiPhi29 DNA Polymerase is a proprietary phi29 DNA Polymerase mutant developed through in vitro protein evolution. This enzyme is significantly improved over phi29 DNA Polymerase in protein thermostability, reaction speed, product yield, and amplification bias. At the same time, it retains all the benefits of the wild-type enzyme, including high processivity (>70 kb), strong strand-displacing activity, and 3΄ to 5΄ exonuclease (proofreading) activity. EquiPhi29 DNA Polymerase is also available as a convenient kit with all required components for RCA and MDA-WGA. Product highlights • Fast—amplifies target in less than 2 hours • Sensitive—provides sensitivity down to 1 fg of DNA • High yield—yields up to 17 μg amplified DNA • Variety of applications—compatible with cell-free DNA enrichment, and cell-free protein expression • Availability—available in lyo-ready format Find out more about EquiPhi29 DNA Polymerase at thermofisher.com/equiphi29. Did you know? These products are free of OPE and NPE, making them safer for aquatic life. Table of contents 59 Isothermal amplification Recombinase polymerase amplification (RPA) RPA is a sensitive, low-temperature (39–42°C) isothermal DNA amplification technique that is initiated with two primers and carried out using a mixture of recombinase enzymes, a single-stranded DNA–binding protein (SSB), and a strand-displacing DNA polymerase such as Bst DNA polymerase. To detect RNA targets, a reverse transcriptase can be incorporated into the RPA reaction. Amplified DNA can be detected using various detection methods such as endpoint detection, real-time analysis, lateral flow assays, and CRISPR-Cas9 systems. As shown below, RPA uses a recombinase complex made of UvsX and UvsY proteins, which binds to the primers in the presence of ATP. This complex seeks a sequence on the DNA homologous to the primer, and promotes strand opening and invasion by the primer. The SSB stabilizes the resulting D-loop structure, allowing the DNA polymerase to extend the primer while displacing the DNA strand. Lyo-ready RPA Kit The Invitrogen™ Lyo-ready RPA Kit includes all required components in a glycerol-free format for isothermal RPA, enabling high reaction sensitivity, specificity, and tolerance to inhibitors. The Lyo-ready RPA Kit contains separate reagents that provide maximum flexibility to optimize the RPA reaction and work with various types of pathogens (RNA or DNA), including S. aureus, P. aeruginosa, influenza and measles viruses, and others. Components for RPA and RT-RPA reactions are also available in a stand-alone format. Product highlights • Sensitive—provides sensitivity down to as low as one copy of target • Specific—detects and amplifies a single target in a mixture of RNA and DNA • Fast—amplifies target in as little as 20 minutes • Robust—amplifies even from inhibitor-containing RNA and DNA samples Find out more about th Learn more about RPA at thermofisher.com/rpa. e Lyo-ready RPA Kit here. 60 Table of contents Resources Table of contents 61 We have compiled educational resources, mobile apps, frequently asked questions, and other information to help you achieve success in your research. 62 Table of contents Reverse transcription Restriction enzymes, molecular cloning, and competent cells PCR enzymes, plastics, and thermal cyclers Nucleic acid electrophoresis Educational resources Suitable for new and experienced molecular biologists alike, our free online educational resources are designed to help you review the basics, build your expertise, or discover our latest innovative technologies. Explore our educational resources in the following areas of molecular biology. Resources Resources Webinars: Watch live and recorded webinars for in-depth understanding of molecular and synthetic biology techniques and tools to help elevate your research. thermofisher.com/mbwebinars Videos: Experience entertaining and visual learning with our educational videos on molecular biology techniques, how-to guides, tips and tricks, and more. thermofisher.com/mbvideos Application notes: Read white papers and application notes from our R&D scientists on our product innovations. thermofisher.com/mbliterature Online tools: Use our interactive online tools for PCR annealing temperature, restriction enzyme information, product selection, and more. thermofisher.com/mbtools Find out more at thermofisher.com/mbschool thermofisher.com/rteducation thermofisher.com/pcreducation thermofisher.com/na-electrophoresis-education thermofisher.com/cloningeducation Table of contents 63 As a leading supplier of molecular biology products, we offer tailored solutions for companies developing new molecular assays. Whether you’re just starting or need a specific solution, we have what you need. Work with an experienced supplier that knows both raw materials and new technologies. Our dedicated business team is here to provide value beyond just our products. What do our OEM solutions mean to you? Choosing greener products? Look for the leaf. For customers seeking greener products, Thermo Fisher has created an easy identification system with our green leaf symbol. When you see the green leaf symbol on one of our products, it means that product meets one or more of our environmental sustainability criteria. Mobile apps Podcast: Speaking of Mol Bio Custom Commercial Supply PCR Quest—match-3 lab game Test your PCR knowledge with our lab game—PCR Quest—where you travel from lab to lab crushing the world’s toughest diseases. Download at thermofisher.com/pcrquest. DailyCalcs—science calculator The DailyCalcs app turns your phone into a science calculator to help simplify everyday tasks in the lab. The app features eight calculators: molarity, dilution, formula weight, transfection, unit conversions, culture vessel data, media conversions, and specific productivity. Find it in your preferred mobile application store. Listen now to Speaking of Mol Bio. This podcast highlights trending applications in science and the molecular biological aspects of those applications. Our hosts delve into deep discussions with CEOs, R&D scientists, researchers, and key opinion leaders across the globe. This podcast helps scientifically curious people—from all scientific and nonscientific backgrounds—understand how modern molecular biology applications can help push the boundaries in medicine, science, drug discovery, and in the cure and treatment of diseases. Listen now at thermofisher.com/molbiopodcast. Instrument Connect—remote monitoring Instrument Connect allows you to view instrument status, monitor or schedule a run, and more on any cloud-enabled instrument, including the Applied Biosystems™ ProFlex™, SimpliAmp™, and MiniAmp™ PCR instruments. Access at thermofisher.com/connect. Find out more at thermofisher.com/mdx Security of supply Value beyond the product Partnership Workflow solutions Access to commercial rights Resources and expertise Quality Customization End of life Product use and maintenance Manufacturing Packaging and transport Product design and development Sourcing and procurement Resources • Customization of products and services • Consultation, partnership, and expertise • Negotiated business terms • Warranties and indemnification • Commercial-use rights and obligations • Risk and liability management 64 Table of contents Sample preparation Which kit should I use to isolate nucleic acids from my sample? Choosing the right product is fundamental to ensuring proper lysis of cells and tissue, as well as sufficient yield and quality of isolated nucleic acids. Look to our selection guides (see pages 11–14) to help you decide according to nucleic acid type, sample source, experimental throughput, and format as well as downstream applications. What are the key steps to preventing RNA degradation? The basic lab precautions listed below can help minimize RNA degradation and avoid experimental inconsistency and failure. • Use nuclease-free pipette tips and tubes • Use nuclease-free water and reagents • Regularly decontaminate work surfaces • Properly stabilize RNA sources before storage For more tips and troubleshooting advice on sample prep, visit thermofisher.com/rnabasics and thermofisher.com/napsupport. Reverse transcription How do I improve the efficiency of cDNA synthesis when working with challenging samples (e.g., low-abundance, degraded, inhibitor-containing, or GC-rich RNA)? When working with challenging RNA samples, select a reverse transcriptase that is highly sensitive, processive, thermostable, and resistant to common inhibitors, to help you obtain the highest cDNA yield (see pages 17–19). Frequently asked questions Below are some common questions and answers to help you start or troubleshoot molecular biology experiments. What are the benefits of using random primers, oligo(dT) primers, gene-specific primers, or oligo(dT)/random mixed primers in reverse transcription? • Random primers are good to use with degraded RNA, RNA with high secondary structure, nonpolyadenylated RNA, or prokaryotic RNA. • Oligo(dT) primers are an optimal choice for synthesis of full-length cDNA from eukaryotic mRNA. Applications include cDNA cloning, cDNA library construction, and 3´ rapid amplification of cDNA ends (3´ RACE). • Gene-specific primers are designed based on known sequences of the target RNA. These primers offer the most specific priming and are commonly used in one-step RT-PCR. • A mixture of oligo(dT) and random primers is often used in two-step RT-PCR to achieve the benefits of each primer type (see page 22). For more tips and troubleshooting advice on reverse transcription, visit thermofisher.com/rteducation. PCR amplification How can I optimize primer annealing for PCR? Traditionally, gradient thermal cyclers have been used to simultaneously assess multiple temperatures around the theoretical annealing point. Compared to gradient thermal cyclers, instruments with VeriFlex technology allow more precise temperature control for faster optimization of primer annealing (see pages 26–27). Tedious optimization steps may be circumvented using the novel Invitrogen™ Platinum™ DNA polymerases. Their innovative buffers enable specific annealing at 60°C for most primers when they are designed following general primer design rules (see pages 30–33). Resources Table of contents 65 What do I need to run fast PCR? PCR amplicons shorter than 1 kb can be amplified in as little as 40 minutes using “fast” enzymes (high processivity; see page 32), “fast” plastics (low profile and ultra-thin walls; see page 29), and “fast” thermal cyclers (fast ramp rate; see pages 26–27). How can I prevent sample evaporation during PCR? Proper sealing of your reactions will help prevent evaporation during PCR. • When using adhesive film to seal a plate, be sure to properly align the seal to cover all wells and press firmly along all edges of the plate using an applicator tool. • When sealing a plate using cap strips, ensure that the cap strips are compatible with the plate and thermal cycler being used. Be sure to align cap strips with each well of the plate and place firmly across the plate for a secure fit. • Use the applicator tool (Cat. No. 4333183 or 4330015) or other comparable sealing tools as needed. For more tips and troubleshooting advice on PCR, visit thermofisher.com/pcreducation and thermofisher.com/pcrsupport. Frequently asked questions (cont.) Nucleic acid electrophoresis Why is it important to choose the right ladder when using E-Gel precast agarose gels? Accurate analysis of electrophoresis bands often depends on the DNA ladder chosen for your gel run. E-Gel DNA ladders are formulated with ready-to-use buffers unique for E-Gel precast agarose gels, and DNA standards designed for optimal separation (see page 42). Are there safer alternatives to ethidium bromide for staining nucleic acids in gel electrophoresis? SYBR Safe DNA Gel Stain is a safer alternative to ethidium bromide and is commonly used in gel electrophoresis. SYBR Safe DNA stain is not classified as hazardous waste or as a pollutant under US federal regulations (see page 42). Do I need a buffer to run the E-Gel system? No. The E-Gel electrophoresis system does not require electrophoresis buffers like TBE or TAE. E-Gel cassettes already contain everything you will need and are classified as dry electrophoresis. For more tips and troubleshooting advice on nucleic acid electrophoresis, visit thermofisher.com/na-electrophoresis-education and thermofisher.com/na-electrophoresis-support. Cloning Do you have a buffer compatibility chart for restriction enzymes? All FastDigest restriction enzymes are 100% active in one universal FastDigest buffer (see page 46). Hence, there is no buffer compatibility chart for FastDigest restriction enzymes. What is the main difference between GeneArt Strings DNA Fragments and GeneArt Gene Synthesis? GeneArt Strings DNA Fragments are custom-made, linear DNA fragments that are uncloned and double-stranded. GeneArt Gene Synthesis is a service offered for chemical synthesis, cloning, and sequence verification of genetic sequences (see page 50). What are some key considerations for choosing competent cells for my cloning applications? Genotype, transformation efficiency, growth rate, and throughput format are important factors in choosing competent cells for cloning. The genotype of a cell strain may determine growth conditions and suitability for transformation with specific DNA types (see page 51). For more tips and troubleshooting advice on cloning, visit thermofisher.com/cloningeducation and thermofisher.com/cloningsupport. Resources 66 Table of contents Quantity Cat. No. Nucleic acid isolation PureLink Quick Plasmid Miniprep Kit 50 preps K210010 PureLink HiPure Plasmid Filter Midiprep Kit 25 preps K210014 PureLink HiPure Plasmid Filter Maxiprep Kit 10 preps K210006 PureLink Pro Quick96 Plasmid Purification Kit 4 x 96 preps K211004A PureLink Quick Gel Extraction Kit 50 preps K210012 TRIzol Plus RNA Purification Kit 50 preps 12183555 PureLink RNA Mini Kit 10 preps 12183020 PureLink Genomic DNA Mini Kit 10 preps K182000 PureLink Pro 96 Genomic DNA Purification Kit 4 x 96 preps K182104A PureLink Pro 96 Viral RNA/DNA Purification Kit 4 plates 12280096A PureLink Viral RNA/DNA Mini Kit 50 preps 12280050 PureLink Genomic Plant DNA Purification Kit 50 preps K183001 MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, with bead tubes 100 preps A42358 MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, with bead plate 100 preps A42357 MagMAX Cell-Free DNA Isolation Kit 1 kit A29319 MagMAX Cell-Free Total Nucleic Acid Isolation 1 kit A36716 MagMAX FFPE DNA/RNA Ultra Kit 1 kit A31881 MagMAX mirVana Total RNA Isolation Kit 96 reactions A27828 MagMAX Sequential DNA/RNA Kit 1 kit A65309 MagMAX Prime Viral/Pathogen NA Isolation Kit Up to 600 reactions A58145 KingFisher PlasmidPro Maxi Processor Endotoxin-Free Cartridge 4 cartridges A54072 PureLink PCR Purification Kit 50 preps K310001 PureLink Quick Gel Extraction and PCR Purification Combo Kit 50 preps K220001 PureLink Quick Gel Extraction Kit 50 preps K210012 MagMAX Pure Bind Beads 5 mL A58521 50 mL A58522 250 mL A58523 TaqMan Fast Advanced Cells-to-CT Kit 40 reactions A35374 100 reactions A35377 Dynabeads M-270 Streptavidin 2 mL 65305 Dynabeads MyOne Streptavidin C1 2 mL 65001 Quantity Cat. No. KingFisher instruments – For Laboratory Use. KingFisher Apex Purification System with 96 PCR Head 1 system 5400910 KingFisher Apex Purification System with 96 Combi Head 1 system 5400920 KingFisher Apex Purification System with 96 Deep-Well Head 1 system 5400930 KingFisher Apex Purification System with 24 Combi Head 1 system 5400940 KingFisher Flex Purification System with 96 PCR Head 1 system 5400610 KingFisher Flex Purification System with 96 KingFisher Plate 1 system 5400620 KingFisher Flex Purification System with 24 Deep-Well Head 1 system 5400640 KingFisher Flex Purification System with 96 Deep-Well Head 1 system 5400630 KingFisher Presto Purification System with 24 Deep-Well Head 1 system 5400840 KingFisher Presto Purification System with 96 Deep-Well Head 1 system 5400830 KingFisher instruments – For Research Use Only. Not for use in diagnostic procedures. KingFisher Duo Prime Purification System 1 system 5400110 KingFisher PlasmidPro Maxi Processor 1 instrument A66427 Ordering information Resources Table of contents 67 Ordering information (cont.) Resources Quantity Cat. No. Reverse transcription SuperScript IV Reverse Transcriptase 2,000 units 18090010 10,000 units 18090050 SuperScript IV First-Strand Synthesis System 50 reactions 18091050 200 reactions 18091200 SuperScript IV VILO Master Mix 50 reactions 11756050 500 reactions 11756500 SuperScript IV VILO Master Mix with ezDNase Enzyme 50 reactions 11766050 500 reactions 11766500 SuperScript IV One-Step RT-PCR System 25 reactions 12594025 100 reactions 12594100 SuperScript IV UniPrime One-Step RT-PCR System 100 reactions 12597100 SuperScript IV CellsDirect cDNA Synthesis Kit 50 reactions 11750150 500 reactions 11750350 SuperScript IV CellsDirect Lysis Reagents 500 reactions 11750550 SuperScript IV Single Cell/Low Input cDNA PreAmp Kit 48 reactions 11752048 96 reactions 11752096 192 reactions 11752192 SuperScript IV Template Switching RT Master Mix 48 reactions A65423 96 reactions A65424 5 x 96 Reactions A65425 SUPERase·In RNase Inhibitor (20 U/μL) 2,500 units AM2694 10,000 units AM2696 RNaseOUT Recombinant Ribonuclease Inhibitor 5,000 units 10777019 RNase Inhibitor 2,000 units N8080119 Ambion RNase Inhibitor, cloned, 40 U/μL 2,500 units AM2682 10,000 units AM2684 Ribonuclease H 30 units 18021014 Random Hexamers (50 μM) 5 nmol N8080127 Quantity Cat. No. Random Primers 100 μL 48190011 Oligo(dT)12–18 Primer 50 μL 18418012 Oligo(dT)20 Primer 50 μL 18418020 DNase I, Amplification Grade 100 units 18068015 Isothermal amplification Lyo-ready Bst DNA Polymerase 1,200 units (6 U/μL) A56655 6,000 units (6 U/μL) A56656 1,200 units (40 U/μL) A56657 SuperScript IV RT-LAMP Master Mix 100 reactions A51801 400 reactions A51802 1,000 reactions A51803 EquiPhi29 DNA Polymerase 250 units A39390 1,000 units A39391 5,000 units A39392 EquiPhi29 DNA Amplification Kit 100 reactions A65393 500 reactions A65394 phi29 DNA Polymerase (10 U/μL) 250 units EP0091 1,000 units EP0092 5,000 units EP0094 Lyo-ready RPA Kit 100 reactions A72127 500 reactions A72128 Lyo-ready T4 UvsX Protein 120 μg A72124 Lyo-ready T4 UvsY Protein 120 μg A72125 Lyo-ready T4 Gene 32 Protein 1,600 μg A72123 L.b. Cas12a Nuclease 200 pmol A40001796 2,000 pmol A40001795 68 Table of contents Quantity Cat. No. PCR Thermal cyclers ProFlex PCR System, 3 x 32-well 1 instrument 4484073 ProFlex PCR System, 96-well 1 instrument 4484075 VeritiPro Thermal Cycler, 96-well 1 instrument A48141 MiniAmp Plus Thermal Cycler 1 instrument A37835 MiniAmp Thermal Cycler 1 instrument A37834 Automated Thermal Cycler (ATC), 96-well, laptop 1 instrument A31486 Plastics MicroAmp EnduraPlate Optical 96-Well Fast Multicolor Reaction Plates with Barcode 5 plates 4483493 MicroAmp Optical Adhesive Film 100 covers 4311971 MicroAmp Optical 96-Well Reaction Plate 10 plates N8010560 MicroAmp Optical 8-Cap Strips 300 strips 4323032 MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL 10 plates 4346907 MicroAmp Fast Reaction Tube with Cap, 0.1 mL 1,000 tubes 4358297 MicroAmp EnduraPlate Optical 384-Well Multicolor Reaction Plates with Barcode 5 plates 4483316 MicroAmp EnduraPlate Optical 96-Well Clear Reaction Plates with Barcode 20 plates 4483354 MicroAmp TriFlex 3 x 32-Well PCR Reaction Plate 20 plates A32811 MicroAmp 8-Tube Strip with Attached Domed Caps, 0.2 mL 125 strips A30589 MicroAmp EnduraPlate Optical 96-Well Full-Skirted Plates with Barcode, clear 50 plates A31728 PCR Plate, 384-well, standard, Sustain Series 50 plates AB1384SS PCR Plate, 96-well, non-skirted, Sustain Series 25 plates AB0600SS PCR Plate, 96-well, segmented, semi-skirted, Sustain Series 25 plates AB0900SS Tubes and Flat Caps, strips of 8, Sustain Series 250 strips AB1182SS Quantity Cat. No. PCR enzymes Platinum II Taq Hot-Start DNA Polymerase 100 reactions 14966001 500 reactions 14966005 Platinum II Hot-Start PCR Master Mix (2X) 50 reactions 14000012 200 reactions 14000013 Platinum II Hot-Start Green PCR Master Mix (2X) 50 reactions 14001012 200 reactions 14001013 AmpliTaq Gold 360 DNA Polymerase 100 units 4398813 250 units 4398823 AmpliTaq Gold 360 Master Mix 1 mL 4398876 5 mL 4398881 Platinum SuperFi II DNA Polymerase 100 units 12361010 500 units 12361050 Platinum SuperFi II PCR Master Mix 100 reactions 12368010 500 reactions 12368050 Platinum SuperFi II Green PCR Master Mix 100 reactions 12369010 500 reactions 12369050 Platinum Direct PCR Universal Master Mix 100 reactions A44647100 500 reactions A44647500 dNTP Set (100 mM) 4 x 250 μL 10297018 8 x 1.25 mL 10297117 Ordering information (cont.) Resources Order custom DNA oligos at thermofisher.com/orderoligos Table of contents 69 Quantity Cat. No. Cloning and gene synthesis FastDigest BamHI 800 reactions FD0054 2,500 reactions FD0055 FastDigest BcuI 20 reactions FD1253 50 reactions FD1254 FastDigest BshTI 20 reactions FD1464 FastDigest DpnI 50 reactions FD1703 100 reactions FD1704 FastDigest EcoRI 800 reactions FD0274 2,500 reactions FD0275 FastDigest KpnI 300 reactions FD0524 FastDigest NotI 20 reactions FD0593 50 reactions FD0594 150 reactions FD0595 250 reactions FD0596 FastDigest SaII 200 reactions FD0644 FastDigest XbaI 300 reactions FD0684 750 reactions FD0685 FastDigest XhoI 400 reactions FD0694 1,200 reactions FD0695 FastDigest Esp3I (BsmBI) (IIS class) 20 reactions FD0454 FastDigest BpiI (BbsI) (IIS class) 20 reactions FD1014 FastDigest Eco31I (BsaI) (IIS class) 50 reactions FD0293 100 reactions FD0294 T4 DNA Ligase (5 U/μL) 200 units EL0014 1,000 units EL0011 FastAP Thermosensitive Alkaline Phosphatase (1 U/μL) 300 units EF0654 1,000 units EF0651 Resources Ordering information (cont.) Quantity Cat. No. Nucleic acid separation and analysis SYBR Safe DNA Gel Stain 400 μL S33102 SYBR Gold Nucleic Acid Gel Stain 500 μL S11494 UltraPure DNase/RNase-Free Distilled Water 500 mL 10977015 UltraPure Agarose 100 g 16500100 TrackIt 100 bp DNA Ladder 100 applications 10488058 UltraPure TAE Buffer, 10X 4 L 15558026 E-Gel Agarose Gels with SYBR Safe DNA Gel Stain 10 gels A42135 E-Gel Double Comb Agarose Gels with SYBR Safe DNA Gel Stain 10 gels A42348 E-Gel EX Double Comb Agarose 10 gels A44889 E-Gel CloneWell II Agarose Gels with SYBR Safe DNA Gel Stain, 0.8% 10 gels G661818 E-Gel Agarose Gels with SYBR Safe DNA Gel Stain, 2% 2 x 10 gels A45204 E-Gel EX Agarose Gels, 2%, with SYBR Gold DNA Stain 20 gels G402022 E-Gel 1 Kb Plus DNA Ladder 100 applications 10488090 E-Gel Sample Loading Buffer, 1X 4 x 1.25 mL 10482055 E-Gel Power Snap Plus Electrophoresis System 1 system G9301* G9311** E-Gel Power Snap Plus Electrophoresis System Starter Kit, 48-well, 1% 1 kit G9341* G9331** E-Gel Power Snap Plus Electrophoresis System Starter Kit, 48-well, 2% 1 kit G9342* G9332** E-Gel Power Snap Plus Electrophoresis System Starter Kit, 96-well, 1% 1 kit G9391* G9381** E-Gel Power Snap Plus Electrophoresis System Starter Kit, 96-well, 2% 1 kit G9392* G9382** E-Gel Power Snap Electrophoresis System Starter Kit, EX 2% 1 kit G8342ST E-Gel 48 Agarose Gels with SYBR Safe DNA Gel Stain, 2% 8 gels G820802 E-Gel 96 Agarose Gels with SYBR Safe DNA Gel Stain, 2% 8 gels G720802 E-Gel 48 Agarose Gels with SYBR Safe DNA Gel Stain, 4% 8 gels G820804 * North America, Europe, Middle East, and Africa. ** Asia Pacific, Japan, Latin America, and greater China. 70 Table of contents Order custom cloned genes or DNA fragments at thermofisher.com/geneartdashboard. Resources Quantity Cat. No. T4 Polynucleotide Kinase (10 U/μL) 500 units EK0031 2,500 units EK0032 DNA Polymerase I (10 U/μL) 500 units EP0041 2,500 units EP0042 Klenow Fragment (10 U/μL) 300 units EP0051 1,500 units EP0052 T4 DNA Polymerase (5 U/μL) 1 mL of 5X reaction buffer EP0061 2 x 1 mL of 5X reaction buffer EP0062 T7 DNA Polymerase (10 U/μL) 300 units EP0081 CorrectASE Enzyme 50 reactions A14972 TOPO TA Cloning Kit for Subcloning, without competent cells 25 reactions 450641 Zero Blunt TOPO PCR Cloning Kit, without competent cells 25 reactions 450245 pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli 20 reactions K240020 One Shot TOP10 Chemically Competent E. coli 21 x 50 μL C404003 One Shot Stbl3 Chemically Competent E. coli 21 x 50 μL/tube C737303 MAX Efficiency DH5α Competent Cells 5 x 200 μL 18258012 ElectroMAX DH10B Cells 5 x 100 μL 18290015 MAX Efficiency Stbl2 Competent Cells 5 x 200 μL 10268019 MultiShot TOP10 Chemically Competent E. coli 5 plates C40005 MultiShot StripWell TOP10 Chemically Competent E. coli 1 rack C409601 MultiShot StripWell BL21 Star (DE3) Chemically Competent E. coli 1 rack C609601 MultiShot FlexPlate TOP10 Chemically Competent E. coli 1 plate C4081201 MultiShot FlexPlate DH5α T1R Chemically Competent E. coli 1 plate C4481201 MultiShot FlexPlate Stbl3 Chemically Competent E. coli 1 plate C7381201 Ordering information (cont.) Quantity Cat. No. GeneArt Gibson Assembly HiFi Master Mix 50 reactions A46628 GeneArt Gibson Assembly EX Master Mix 50 reactions A46636 GeneArt Seamless Cloning and Assembly Enzyme Mix 20 reactions A14606 GeneArt Type IIS Assembly Kit, AarI 10 reactions A15916 GeneArt Type IIS Assembly Kit, BsaI 10 reactions A15917 GeneArt Type IIS Assembly Kit, BbsI 10 reactions A15918 GeneArt High-Order Genetic Assembly System 10 reactions A13285 Gateway BP Clonase II Enzyme Mix 20 reactions 11789020 Gateway LR Clonase II Enzyme Mix 20 reactions 11791020 MultiSite Gateway Pro Plus 20 reactions 12537100 LR Clonase II Plus Enzyme 20 reactions 12538120 Gateway Vector Conversion System with One Shot ccdB Survival Cells 1 kit 11828029 PCR Cloning System with Gateway Technology with pDONR 221 and OmniMAX 2 Competent Cells 20 reactions 12535029 PCR Cloning System with Gateway Technology with pDONR/Zeo and OmniMAX 2 Competent Cells 20 reactions 12535037 Gateway pDONR 221 Vector 6 μg 12536017 pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli 20 reactions K240020 pCR 8/GW/TOPO TA Cloning Kit with One Shot TOP10 E. coli 20 reactions K250020 Table of contents 71 For Research Use Only. Not for use in diagnostic procedures. © 2020–2023, 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan and AmpliTaq Gold are trademarks of Roche Molecular Systems, Inc., used under permission and license. TRIzol and DNAzol are trademarks of Molecular Research Center, Inc. Gibson Assembly is a trademark of Telesis Bio, Inc., used under permission and license. BROC-9207299 0425 Find out more at thermofisher.com/molecularmasterpiece