In-Fusion HD Cloning Plus System

Transcription

1 In-Fusion HD Cloning Plus System One trustworthy solution for all your cloning and mutagenesis projects Seamless Directional Any vector GOI + Any insert Anywhere Large & small inserts or vectors Point mutations Multiple fragments Build your own vector One GOI different vectors One vector different inserts Libraries

2 In-Fusion HD Cloning Plus The Only System you need Single-tube protocol x x Recombinant vector Amplify your gene of interest PCR product Gene-specific primers with 15 bp extensions homologous to vector ends Any linearized vector The In-Fusion enzyme produces single-stranded PCR fragment and vector ends that are fused due to the 15 bp homology Simple, versatile and restriction-site independent Our In-Fusion HD Cloning Plus System facilitates the directional cloning of any oligo/pcr fragment (20 bp to 15 kb) or multiple fragments into any linearized vector in a single reaction. The method relies on our stable proprietary In-Fusion HD enzyme that can fuse oligos/pcr-generated sequences to linearized vectors efficiently and precisely by recognizing a 15 bp overlap at their ends. This system allows sequence and restriction site-independent cloning: clone any insert, into any location, within any No vector dephosphorylation, blunt-end polishing, or PCR fragment digestion is required.moreover, the linearized vector can also be generated by inverse PCR instead of restriction digestion (1). How does In-Fusion work? The In-Fusion HD Cloning Plus System functions through a ligase-independent mechanism. Firstly, the In-Fusion enzyme creates single-stranded regions at the ends of the oligo/pcr insert and linearized This exposes the 15 bp complementary regions on the insert and vector DNA molecules that then spontaneously anneal/fuse through base pairing. Introduction of this annealed/fused DNA into Escherichia coli repairs any single-stranded gaps and results in the synthesis of a recombinant vector containing the oligo/pcr insert. Fast, seamless and superior cloning efficiency A simple minute reaction results in the creation of seamless and precisely engineered constructs, where no extra bp of vector or restriction-site-derived DNA is added. This is a crucial advantage when generating constructs to express tagged proteins as no undesirable extra amino acids are added to the protein. In-Fusion HD Cloning Plus allows you to achieve >95% cloning efficiency, even when cloning large fragments or multiple fragments. Clone anything from small oligos to large fragments The In-Fusion HD Cloning Plus System enables the directional cloning of almost any sized fragment ranging from 20 bp oligos (excluding 15 bp overlaps) to large 15 kb PCR fragments into any linearized vector in a single reaction. This way, you do not waste any valuable time optimising the cloning of small or large fragments using traditional cloning methods. Colony number 3,000 2,000 1,000 9/10 correct clones 10/10 correct clones 10/10 correct clones Fragment size (kb) Ten out of ten colonies contain the correct insert (100% efficiency) when cloning fragments as large as 15 kb (results confirmed by colony PCR screening).

3 3 fragments 4 fragments Total colonies: 415 Correct clones: 7/10 Total colonies: 155 Correct clones: 8/10 Using this system, cloning up to four fragments simultaneously is as easy as cloning a single fragment. Just combine the PCR fragments with appropriate complementary ends and a linear vector, then incubate. This saves weeks that would otherwise be spent screening clones and subcloning. Design primers for each fragment that you are cloning into the When designing primers, include 15 bp of overlapping sequence with the adjacent segment or linearized Amplify your fragments for cloning using the appropriately designed primers. Simultaneous cloning of many different inserts into one vector Save time and money by using In-Fusion to simultaneously clone in parallel, many different inserts into your vector of choice, in a single reaction. For example, Zhou et al. utilised In-Fusion cloning to assist in the rapid generation of recombinant influenza A viruses that could then be used as vaccine seed stocks (2). Specifically, eight genomic segments amplified by multi-segment PCR directly from human swab specimens were cloned simultaneously into the StuI-linearized pg26a12 vector in a single In-Fusion cloning reaction (2). Simultaneous cloning of one insert into multiple vectors Conversely, In-Fusion can be used to clone your insert of choice simultaneously into multiple vectors, in a single cloning operation. For example, Berrow et al. generated the popin protein expression vector set where several vectors within the series were engineered at the site of linearization to enable the same PCR product to be cloned into them all in parallel via In-Fusion (9). In this way, In-Fusion was utilised to generate in parallel multiple expression vectors containing the same insert but with different tags, thus saving a lot of time and effort. In addition, this study also illustrates the cost-effective use of PCR primers. Clone multiple fragments with ease! Using our In-Fusion HD Cloning Plus system, cloning multiple fragments simultaneously is as easy as cloning a single fragment and it can be done in a single reaction! Just combine the PCR fragments with appropriate complementary ends and a linear vector, then incubate with the In-Fusion enzyme. This saves weeks and months that would otherwise be spent screening clones and sub-cloning. The ability to easily, rapidly and precisely clone many fragments at once will help to speed up the generation of complex target constructs in your lab. And don t just take our word for it; Zhu et al. successfully constructed a three domain immunoglobulin fusion protein by means of a four-piece In-Fusion reaction (3). In-Fusion BioBrick Assembly Genetic circuits and artificial biological systems can be created by the assembly of modular genetic components or biological parts to form more complex systems. The BioBrick assembly standard was the first attempt to standardize biological parts to allow a single assembly method and it has become the basis for many similar formats (4 7). Examples of BioBricks include promoters, coding sequences and transcriptional terminators. Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. However, the In-Fusion BioBrick Assembly strategy (8) provides an alternative platform for two or more PCRamplified BioBricks to be assembled and re-engineered simultaneously using the In-Fusion PCR Cloning Kit. The In-Fusion approach is faster, more flexible, and has a higher success rate than standard assembly (8). Site-directed mutagenesis Our In-Fusion HD Cloning Plus System can be used for both cloning and mutagenesis, eliminating the need for a separate kit for mutagenesis. Save money, time, and space in your laboratory by getting this one universal system for genetic manipulation which allows you to engineer both single and multiple mutations quickly and efficiently in one reaction.this is achieved through the use of mutagenic primers in the initial PCR step (3). Please visit our website under the section : Support > Applications > Mutagenesis > InFusion_HD_Site_Directed_Mutagenesis for further information on this great application of In-Fusion HTP Cloning for use in HTP protein production studies In addition, In-Fusion technology has been successfully adapted for use in High-Throughput (HTP) Cloning (9-10). The Oxford Protein Production Facility (9) has created the popin vector suite which when combined with In-Fusion, delivers a versatile and precise one-step cloning process, that

4 can be readily adapted to high-throughput structural biology studies. A crucial advantage of the In-Fusion system is that it enables the precise engineering of tagged constructs with no undesirable vector or restriction-site-derived amino acids being added to the expressed protein, unless specifically required by you (9). This allows the expressed tagged protein to mimic the native protein as closely as possible, in order to aid its correct folding, especially important in studies of protein function. 5' XXX SMARTer II A oligonucleotide 5' poly A 3' CDS Primer II A SMARTScribe RT reaction and ds cdna amplification by LD PCR or primer extension Enriched full-length SMARTer ds cdna with known sequence at each end SMARTer cdna synthesis Construct cdna libraries The In-Fusion SMARTer Directional cdna Library Construction Kit provides an easy and dependable method for producing high-quality, full-length cdna libraries as compared to traditional protocols. The kit utilizes two of Clontech s most innovative technologies: SMARTer cdna Synthesis and In-Fusion HD Cloning Plus. SMART (Switching Mechanism At 5 end of RNA Template) cdna synthesis technology is a PCR-based method for cdna library construction that is designed to preferentially enrich for full-length cdnas (11-12). This unique technology allows the efficient incorporation of synthetic adaptors on both the 5 and 3 ends of cdna during first-strand synthesis, without adaptor ligation, in one reverse transcription reaction. In-Fusion cloning makes it easy to clone your SMARTer cdna library into any location within any Clones isolated from SMARTer libraries can be transferred directly to any linearized expression vector for functional analysis, without the need for compatible restriction sites. psmart2ifd or any linearized vector Purify/size fractionate cdna Clone cdna library in only 30 minutes using In-Fusion Transform competent E. coli Titrate and amplify cdna library In-Fusion cloning References 1. Benoit R.M. et al. (2006) Protein Expression and Purification 45(1) : Zhou B., et al. (2009) J. Virol. 83(19): Zhu B. et al. (2007) BioTechniques 43(3): Knight, T. F. (2003) DSpace: 5. Shetty, R. P. et al. (2008) J Biol Eng. 2: Phillips, I. E. and Silver, P. A. (2006) DSpace: 7. Anderson, J. C. et al. (2010) J. Biol. Eng. 4(1):2. content/4/1/1 8. Sleight, S. C. et al. (2010) Nucleic Acids Res. 38(8): Berrow, N.S. et al. (2007) Nucleic Acids Res. 35(6):e Marsischky, G. & LaBaer, J. (2004) Genome Res. 14: Zhu, Y. et al. (July 1996) Clontechniques XI(3): Wellenreuther, R. et al. (October 2005) Clontechniques XX(2):24 25.

5 Comparison of PCR cloning methods Advantages of using In-Fusion HD Cloning Plus Limitations of ligationdependent cloning Limitations of TA cloning systems Limitations of other vectordependent cloning systems Flexible Clone ANY PCR fragment into ANY vector in ANY location. Dependent on availability and position of specific restriction sites. Limited to using provided Limited to using specific specialized vectors provided. Multiple fragment cloning Assemble up to five fragments directly into your vector of choice in one single step. Not possible to clone multiple fragments at once into a single Usually need to assemble each fragment sequentially via many subcloning steps. Not possible to clone multiple fragments at once into a single Usually need to assemble each fragment sequentially via many subcloning steps. Possible to clone multiple fragments but requires two steps. One-step cloning Clone directly into your vector of choice. No time consuming sub-cloning steps involved. Sub-cloning steps may be required. Sub-cloning steps may be required. Sub-cloning steps may be required. Directional Obtain clones with your insert in the correct orientation, firsttime round. Blunt-end cloning is generally not very efficient and in some cases you may need to screen many colonies before obtaining positive clones with the insert in the correct orientation. Non-directional cloning which generally requires screening many clones. Highly Efficient Obtain >95% correct clones for fragments across a wide size range, including GC-rich fragments. Generally require repurification of insert after restriction digestion. Need to use a low quality proofreading DNA polymerase or add an A tail. May require the use of specialized competent cells. Seamless Obtain desired construct without the addition of unwanted base pairs of DNA from the cloning Particularly important for creating tagged protein expression/mutant vectors. Positive clones might contain additional unwanted base pairs of DNA from the cloning Positive clones might contain additional unwanted base pairs of DNA from the cloning Positive clones contain additional unwanted base pairs of DNA from the cloning Streamlined The same simple and fast protocol for multiple applications. Restriction digest of both insert and vector usually required. Vector dephosphorylation step generally required to reduce the number of background colonies. Usually a different kit is required for each HTP-Ready format Compatible with robotics and automation. Not suitable for HTP due to the need to screen many colonies before obtaining a positive clone with the insert in the correct orientation. Not suitable for HTP due to the need to screen many colonies before obtaining a positive clone with the insert in the correct orientation.

6 New In-Fusion HD Cloning Plus Systems: The Complete Solution for all Your Cloning Projects In order to make In-Fusion cloning even more convenient and cost-effective, we now offer our In-Fusion HD Cloning Plus Systems which include all the reagents you need to get your clone in just two days. In addition to the In-Fusion enzyme premix, you will receive the PCR enzyme, highly efficient competent cells, and a PCR clean-up or gel extraction kit all in one shipment. Higher-Quality Competent Cells In-Fusion HD Cloning Plus System now includes highquality Stellar Competent Cells, which have a wide range of applications including cdna or genomic DNA library construction. Transformation efficiency (cfu/μg puc19 DNA) 1.0 x x x kb 10 kb 20 kb 1.0 x 10 Stellar >1.0 x 10 8 DH5α >1.0 x 10 8 DH10B >1.0 x 10 9 COA transformation efficiency (cfu/μg puc19 DNA) High Fidelity PCR Premix In-Fusion HD Cloning Plus Systems also include CloneAmp HiFi, a high fidelity polymerase that is the ideal enzyme for PCR cloning. CloneAmp HiFi is conveniently packaged as a PCR premix for simple reaction set up. Amplify up to 10 kb rapidly and virtually free of errors. CloneAmp HiFi High Fidelity Enzyme from Competitor A % % % % 0 Pfu Taq Mutation frequency (%) Product Information Format Product Cat.No Size Purification Kit Included Liquid Liquid Lyophilized In-Fusion HD Cloning Plus In-Fusion HD Cloning Plus CE In-Fusion HD EcoDry Cloning Plus Stellar Competent Cells Included CloneAmp HiFi PCR Premix Included (rxns) rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes rxns Cloning Enhancer Yes Yes rxns Cloning Enhancer Yes Yes rxns Cloning Enhancer Yes Yes rxns Cloning Enhancer Yes Yes rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes rxns Spin Columns Yes Yes Related products Product Cat.No Size CloneAmp HiFi PCR Premix rxns In-Fusion SMARTer Directional cdna Library Construction Kit rxns Takara Bio Europe SAS Clontech Laboratories orders@takara-clontech.eu tech@takara-clontech.eu Europe : +33.(0) Austria : Germany : Switzerland : United Kingdom : Notice to Purchaser Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product s web page at It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. BioBrick is a trademark of The BioBricks Foundation, Inc. In-Fusion, Clontech, the Clontech logo, and all other trademarks are the property of Clontech unless noted otherwise. Certain trademarks may not be registered in all jurisdictions. Clontech is a Takara Bio Company Clontech Laboratories, Inc. This brochure is printed on 60% recycled paper. ZZXBR120413EU