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Molecular Cloning

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Molecular Cloning Now includes NEB® Golden Gate Assembly Kit (BsmBI-HF®v2) Molecular Cloning TECHNICAL GUIDE OVERVIEW TABLE OF CONTENTS 3 Online Tools 4–5 Cloning Workflow Comparison 6 DNA Assembly Molecular Cloning Overview 6 Overview Molecular cloning refers to the process by which recombinant DNA molecules are 6 Product Selection produced and transformed into a host organism, where they are replicated. A molecular 7 Golden Gate Assembly Kits 7 Optimization Tips cloning reaction is usually comprised of two components: 8 Technical Tips for Optimizing 1. The DNA fragment of interest to be replicated. Golden Gate Assembly Reactions 2. A vector/plasmid backbone that contains all the components for replication in the host. 9 NEBuilder® HiFi DNA Assembly 10 Protocol/Optimization Tips 10 Gibson Assembly® DNA of interest, such as a gene, regulatory element(s), operon, etc., is prepared for cloning by either excising it out of the source DNA using restriction enzymes, copying it using 11 Cloning & Mutagenesis PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector 11 NEB PCR Cloning Kit ® is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction 12 Q5 Site-Directed Mutagenesis Kit 12 Protocols/Optimization Tips (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and exists separately from the host’s chromosomal or genomic DNA. By physically joining the 13–24 DNA Preparation DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest 13 Nucleic Acid Purification becomes part of the new recombinant plasmid and is replicated by the host. Plasmid vectors 13 Overview 14 cDNA Synthesis allow the DNA of interest to be copied easily in large amounts, and often provide the 14 Overview necessary control elements to be used to direct transcription and translation of the cloned 15 Restriction Enzyme Digestion DNA. As such, they have become the workhorse for many molecular methods such as 15 Overview protein expression, gene expression studies, and functional analysis of biomolecules. 15 Protocol/Optimization Tips 16–21 Performance Chart During the cloning process, the ends of the DNA of interest and the vector have to be 22 Activity in CutSmart Buffer modified to make them compatible for joining through the action of a DNA ligase, 23 PCR/Amplification recombinase, or an in vivo DNA repair mechanism. These steps typically utilize enzymes 23 Overview such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, 23 Product Selection 24 Protocols/Optimization Tips more recently kits have been developed to simplify and standardize these processes. 25–27 Common DNA End Modifications This technical guide will clarify the differences between the various cloning methods, 25 Overview 25 Phosphorylation identify NEB® products available for each method, and provide expert-tested protocols and ® 25 Protocol/Optimization Tips FAQs to help you troubleshoot your experiments and Clone with Confidence . 25 Dephosphorylation 25 Product Selection 25 Protocol/Optimization Tips 26 Blunting/End-repair 26 Product Selection 26 Protocol/Optimization Tips Visit CloneWithNEB.com 27 A-tailing 27 Product Selection 27 Protocol 28 DNA Ligation 28 Vector and Insert Joining 28 Overview 28 Product Selection 29 Protocol/Optimization Tips 30 Transformation 30 Overview 30 Product Selection 30 Protocol/Optimization Tips 31 DNA Analysis 31 DNA Markers and Ladders 31 Overview 31 Product Selection 32 Getting Started with Molecular Cloning • Technical tips and FAQs 33–34 Traditional Cloning Quick Guide • Videos and animations 35–37 Troubleshooting Guide • Much more... 38–39 Ordering Information 2 CLONING TOOLS Online Tools for Cloning Competitor Cross-Reference Tool NEBcutter® V2.0 Use this tool to select another company's competent cell Identify restriction sites within your DNA sequence product and find out which NEB strain is compatible. using NEBcutter. Choose between Type II and Choose either the product name or catalog number from commercially available Type III enzymes to digest your the available selection, and this tool will identify the DNA. NEBcutter V2.0 indicates cut frequency and recommended NEB product and its advantages. A link to the product methylation sensitivity. page where you can also order the product is provided. NEBioCalculator® DNA Sequences and Maps Tool NEBioCalculator is a collection of calculators and With the DNA Sequences and Maps Tool, find converters that are useful in planning bench experiments the nucleotide sequence files for commonly used in molecular biology laboratories. molecular biology tools, including plasmid, viral and bacteriophage vectors. NEBuilder® Assembly Tool Double Digest Finder NEBuilder Assembly Tool can be used to design Use this tool to guide your reaction buffer selection primers for your NEBuilder HiFi and Gibson Assembly when setting up double-digests, a common timesaving reaction, based on the entered fragment sequences and procedure. Choosing the right buffers will help you to the polymerase being used for amplification. avoid star activity and loss of product. PCR Selection Tool Enzyme Finder Use this tool to help select the right DNA polymerase Use this tool to select restriction enzymes by name, for your PCR setup. Whether your amplicon is long, sequence, overhang or type. Enter your sequence using complex, GC-rich or present in a single copy, the PCR single letter code, and Enzyme Finder will identify the selection tool will identify the perfect DNA polymerase right enzyme for the job. for your reaction. Ligase Fidelity Tools REBASE® Visualize overhang ligation preferences, predict Use this tool as a guide to the ever-changing landscape high-fidelity junction sets, and split DNA sequences of restriction enzymes. REBASE, the Restriction Enzyme to facilitate the design of high-fidelity Golden Gate DataBASE, is a dynamic, curated database of restriction assemblies. enzymes and related proteins. NEB Golden Gate Assembly Tool Tm Calculator Use this tool to assist with in silico DNA construct design Use this tool when designing PCR reaction protocols for Golden Gate DNA assembly. It enables the accurate to help determine the optimal annealing temperature design of primers with appropriate Type IIS restriction for your amplicon. Simply input your DNA polymerase, sites and overlaps, quick import of sequences in many primer concentration and your primer sequence and the formats and export of the final assembly, primers and settings. The Tm Calculator will guide you to successful reaction conditions. latest version (v2.1) also incorporates ligase fidelity information. MOBILE APPS NEBaseChanger® NEBaseChanger can be used to design primers specific NEB Tools for iPhone®, iPad® or Android™ to the mutagenesis experiment you are performing NEB Tools brings New England Biolabs’ most popular web tools ® using the Q5 Site-Directed Mutagenesis Kit. This tool to your iPhone, iPad or Android devices. will also calculate a recommended custom annealing temperature based on the sequence of the primers by taking into • Use Enzyme Finder to select a restriction enzyme by category or recognition account any mismatches. sequence, or search by name to find information on any NEB enzyme. Sort your results so they make sense to you, then email them to your inbox or connect directly to www.neb.com. NEBcloner® • Use Double Digest Finder or NEBcloner to determine buffer and reaction conditions Use this tool to find the right products and protocols for experiments requiring two restriction enzymes. for each step (digestion, end modification, ligation ® ® ™ and transformation) of your next traditional cloning When using either of these tools, look for CutSmart , HF and Time-Saver experiment. Also, find other relevant tools and resources enzymes for the ultimate in convenience. NEB Tools enables quick and easy access to the most requested restriction enzyme information, and allows you to plan your to enable protocol optimization. experiments from anywhere. 3 WORKFLOW COMPARISON Cloning Workflow Comparison The figure below compares the steps for the various cloning methodologies, along with the time needed for each step in the workflows. INSERT PREPARATION VECTOR PREPARATION Multiple cloning site Starting Starting (MCS) Material Material RNA OR OR OR OR cDNA Synthesis Plasmid gDNA PCR products Annealed oligos cDNA Plasmid Traditional Cloning OR PCR Cloning OR Seamless Cloning OR LIC (Ligation OR Recombinational Restriction Enzyme OR PCR (RE Digestion & Ligation) (TA & Blunt-End) (Gene Assembly) Independent Cloning) (Gateway/Creator/Univector) (RE) Digestion RE Digestion PCR PCR PCR PCR RE Digestion PCR DNA DNA Preparation 60 min. (Standard) 90 min. 90 min. 90 min. 90 min. Processing 60 min. (Standard) 2 hr. 5–15 min. (Time-Saver) 5–15 min. (Time-Saver) Clean Up 15 min. P P P dsDNA P P P intermediate P OR OR OR OR Clean Up OR A A A A A A 15 min. P Dephosphorylation Clean Up Clean Up Clean Up Linear vector Blunting 15 min. 15 min. 15 min. (Optional) Dephosphorylation (Optional) T-addition DNA End Phosphorylation Cohesive-End Cohesive-End RE Digest DNA End 10–30 min. 10–30 min. 1.5 hr. Modifications (Optional) Formation by Formation by 60 min. (Standard) Modifications 30 min. 5´ → 3´ exo 3´ → 5´ exo 5–15 min. (Time-Saver) Clean Up 15 min OR P 30 min. 30 min. Gel & Column Purification 75 min. T dsDNA OR OR P T intermediate 2 P P P OR A + A Clean Up P P OR P 15 min. Gel and Column Site-Specific Purification Vector & Insert Recombination Linear vector, 75 min. Ligation Ligation Annealing Joining 60 min. ready for joining 15 min. Occurs simultaneously 30 min. Ligation with previous step Proteinase K Treatment Instant – 15 min. Recombination 10 min. Estimated total time 20 min. – 2 hr., 25 min. 3 hr., 45 min. sites 70 min.** T A A T OR OR Holding vector Assembled vector Endpoint vector Estimated total time* 1 hr., 20 min. – 3 hr. 2 hr. – 2 hr., 30 min. 2 hr., 15 min. 2 hr., 45 min.
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