UPDATE Molecular Cloning 2018/19 TECHNICAL GUIDE OVERVIEW TABLE OF CONTENTS 3 Online Tools 4–8 Cloning & Mutagenesis 4 NEBuilder® HiFi DNA Assembly Molecular Cloning Overview 4 Overview Molecular cloning refers to the process by which recombinant DNA molecules are 5 Protocol/Optimization Tips ® produced and transformed into a host organism, where they are replicated. A molecular 5 Gibson Assembly 6 NEB Golden Gate Assembly cloning reaction is usually comprised of two components: 6 Overview 1. The DNA fragment of interest to be replicated. 7 NEB PCR Cloning Kit 2. A vector/plasmid backbone that contains all the components for replication in the host. 7 Overview/Protocols 8 Q5® Site-Directed Mutagenesis Kit 8 Protocols/Optimization Tips 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 9 DNA Assembly Selection Chart PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector 10–20 DNA Preparation is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction 10 Nucleic Acid Purification (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and 11 cDNA Synthesis exists separately from the host’s chromosomal or genomic DNA. By physically joining the 12 Restriction Enzyme Digestion DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest 12 Protocol 12 Optimization Tips becomes part of the new recombinant plasmid and is replicated by the host. Plasmid vectors 13–18 Performance Chart allow the DNA of interest to be copied easily in large amounts, and often provide the 19 PCR necessary control elements to be used to direct transcription and translation of the cloned 19 Protocols DNA. As such, they have become the workhorse for many molecular methods such as 19 Optimization Tips protein expression, gene expression studies, and functional analysis of biomolecules. 20 Product Selection 21–23 Common DNA End Modifications During the cloning process, the ends of the DNA of interest and the vector have to be 21 Phosphorylation modified to make them compatible for joining through the action of a DNA ligase, 21 Protocol recombinase, or an in vivo DNA repair mechanism. These steps typically utilize enzymes 21 Optimization Tips such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, 21 Dephosphorylation more recently kits have been developed to simplify and standardize these processes. 21 Protocol 21 Optimization Tips 21 Product Selection This technical guide will clarify the differences between the various cloning methods, 22 Blunting/End-repair identify NEB® products available for each method, and provide expert-tested protocols and 22 Protocol FAQs to help you troubleshoot your experiments. 22 Optimization Tips 22 Product Selection 23 A-tailing 23 Protocol 23 Product Selection Visit CloneWithNEB.com 23 Activity in CutSmart® Buffer 24 Vector and Insert Joining 24–25 DNA Ligation 24 Protocol 24 Optimization Tips 25 Product Selection 26 Transformation 26 Protocol 26 Optimization Tips 26 Product Selection 27 DNA Markers & Ladders 27 Product Selection 28–29 Traditional Cloning Quick Guide 30–32 Troubleshooting Guide 33 Cloning Workflow Descriptions 33 Seamless Cloning 34 Traditional Cloning • Technical tips and FAQs 34 PCR Cloning 35 Ligation Independent Cloning (LIC) • Videos and animations 35 Recombinational Cloning • Much more... 36–37 Cloning Workflow Comparison 2 38–39 Ordering Information CLONING TOOLS Online Tools for Cloning DNA Preparation Competitor Cross-Reference Tool NEBioCalculator® Use this tool to select another company's competent cell NEBioCalculator is a collection of calculators and product and find out which NEB strain is compatible. converters that are useful in planning bench experiments Choose either the product name or catalog number from in molecular biology laboratories. the available selection, and this tool will identify the recommended NEB product and its advantages. A link to the product page where you can also order the product is provided. NEBuilder® Assembly Tool NEBuilder Assembly Tool can be used to design DNA Sequences and Maps Tool primers for your Gibson Assembly reaction, based on With the DNA Sequences and Maps Tool, find the entered fragment sequences and the polymerase the nucleotide sequence files for commonly used being used for amplification. molecular biology tools, including plasmid, viral and bacteriophage vectors. PCR Selection Tool Use this tool to help select the right DNA polymerase Double Digest Finder for your PCR setup. Whether your amplicon is long, Use this tool to guide your reaction buffer selection complex, GC-rich or present in a single copy, the PCR when setting up double-digests, a common timesaving selection tool will identify the perfect DNA polymerase procedure. Choosing the right buffers will help you to for your reaction. avoid star activity and loss of product. REBASE® Enzyme Finder Use this tool as a guide to the ever-changing landscape Use this tool to select restriction enzymes by name, of restriction enzymes. REBASE, the Restriction Enzyme sequence, overhang or type. Enter your sequence using DataBASE, is a dynamic, curated database of restriction single letter code, and Enzyme Finder will identify the enzymes and related proteins. right enzyme for the job. Tm Calculator NEB Golden Gate Assembly Tool Use this tool when designing PCR reaction protocols Use this tool to assist with in silico DNA construct design to help determine the optimal annealing temperature for Golden Gate DNA assembly. It enables the accurate for your amplicon. Simply input your DNA polymerase, design of primers with appropriate type IIS restriction primer concentration and your primer sequence and the sites and overlaps, quick import of sequences in many Tm Calculator will guide you to successful reaction conditions. formats and export of the final assembly, primers and settings. MOBILE APPS NEBaseChanger® NEBaseChanger can be used to design primers specific ® ® ™ to the mutagenesis experiment you are performing NEB Tools for iPhone , iPad or Android using the Q5® Site-Directed Mutagenesis Kit. This tool NEB Tools brings New England Biolabs’ most will also calculate a recommended custom annealing popular web tools to your iPhone, iPad or temperature based on the sequence of the primers by taking into Android devices. account any mismatches. • Use Enzyme Finder to select a restriction enzyme by category or recognition sequence, or search by name to NEBcloner® find information on any NEB enzyme. Sort your results so Use this tool to find the right products and protocols they make sense to you, then email them to your inbox or for each step (digestion, end modification, ligation connect directly to www.neb.com. and transformation) of your next traditional cloning experiment. Also, find other relevant tools and resources • Use Double Digest Finder or NEBcloner to determine to enable protocol optimization. buffer and reaction conditions for experiments requiring two restriction enzymes. NEBcutter® V2.0 When using either of these tools, look for CutSmart®, HF® Identify restriction sites within your DNA sequence and Time-Saver™ enzymes for the ultimate in convenience. using NEBcutter. Choose between Type II and NEB Tools enables quick and easy access to the most commercially available Type III enzymes to digest your requested restriction enzyme information, and allows you to DNA. NEBcutter V2.0 indicates cut frequency and plan your experiments from anywhere. methylation sensitivity. 3 CLONING & MUTAGENESIS KITS Cloning & Mutagenesis Kits RECOMMENED PRODUCTS NEBuilder HiFi DNA Assembly Cloning Kit NEBuilder HiFi DNA Assembly (NEB #E5520) NEBuilder HiFi DNA Assembly Master Mix NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA (NEB #E2621) fragments, even those with 5´- and 3´-end mismatches. Available with and NEBuilder HiFi DNA Assembly Bundle for without competent E. coli, this flexible kit enables simple and fast seamless Large Fragments cloning utilizing a new proprietary high-fidelity polymerase. Make NEBuilder (NEB #E2623) HiFi your first choice for DNA assembly and cloning. • Simple and fast seamless cloning in as little as 15 minutes • Use one system for both "standard-size" cloning and larger Overview of the NEBuilder HiFi DNA Assembly cloning method gene assembly products (up to 12 fragments and 20 kb) • DNA can be used immediately for transformation or as template for PCR or RCA DNA Preparation • Adapts easily for multiple DNA manipulations, Seamless Cloning From: C B including site-directed mutagenesis • PCR Linear A • Restriction enzyme vector + • Enjoy less screening/re-sequencing of constructs, digestion DNA inserts with 15-30 bp with virtually error-free, high-fidelity assembly • Synthetic DNA overlapping ends (PCR-amplified) (e.g., gBlocks) • Use NEBuilder HiFi in successive rounds of assembly, as it removes 5´- and 3´-end mismatches NEBuilder HiFi DNA Assembly Master Mix • Bridge two ds-fragments with a synthetic ssDNA oligo B for simple and fast construction (e.g., linker insertion A C Single-tube reaction or gRNA library) • Exonuclease chews back 5´ ends to create Assembled • No licensing fee requirements from NEB for single-stranded 3´ overhangs DNA NEBuilder products • DNA polymerase fills in gaps within each annealed fragment Incubate at 50°C • NEBuilder HiFi DNA Assembly Cloning