The gateway cloning system, invented and commercialized by invitrogen since the late 1990s, is a molecular biology method that enables researchers to efficiently transfer dnafragments between plasmids using a proprietary set of recombination sequences, the gateway att sites, and two proprietary enzyme mixes, called lr clonase, and bp. Jun 19, 20 the gateway cloning system, invented and commercialized by invitrogen since the late 1990s, is a molecular biology method that enables researchers to efficiently transfer dnafragments between. Gateway pentr vectors version a 051602 250521 a limited label license covers this product see purchaser notification. Correct design of attb primers for amplification, cloning and expression of a gene in gateway requires consideration of the proper placement of protein expression elements ribosome recognition. Gateway cloning technique allows transfer of dna fragments between different cloning vectors while maintaining the reading frame. For updated protocols and guidelines on using the golden gateway plasmids, please visit golden gateway cloning by stephan kirchmaier. Subcloning is a basic procedure in molecular biology for transfer of dna inserts from one vector to another to gain functionality to study the sequence of interest. Restriction enzyme gateway topo gibson type iis ligation independent cloning oligo stitching molecular cloning or the creation of recombinant dna is an essential process used in scientific. Essentially all subcloning reactions proceed the same way as illustrated in the figure below. Its no wonder gateway cloning has been the goto choice for years, by researchers with varying experiencefrom beginners to advancedfor protein expression, functional analysis, and much more. Its no wonder gateway cloning has been the goto choice for years, by researchers with varying. The gateway technology is a universal cloning method based on the sitespecific recombination properties of bacteriophage lambda landy, 1989.
The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections. Do not use more than 5 l of diluted reaction mix for 50 l competent cells. Basic protocol 1 describes the amplification of target genes and addition of the required recombination sites by pcr, basic protocols 2 and 3 describe the generation of entry master clones, and basic. Cloning kits fiveminute, directional topo cloning of bluntend pcr products into an entry vector for the gateway system catalog numbers k240020, k242020, k252520, k253520, k243520, and k263520 revision date 29 march 2012 publication part number 250434 man0000245. Onestep sequence and ligationindependent cloning as a. The gatewayz technology gatewayz is a universal cloning technology that takes advantage of the sitespecific recombination properties of bacteriophage lambda landy, 1989 to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. Inserts are usually pcr amplified and vectors are made linear either by restriction enzyme digestion or by pcr. Refer to the diagram below and examples 1 and 2, next page for more help.
Both organisms have specific recombination sites called att p in phage l site and att b in e. To convert your cloning vector to a gateway destination vector, you will. Tutorial gateway cloning 2 gateway cloning this tutorial will show you how to use the workbench tool for in silico gateway r cloning. By use of this product, you accept the terms and conditions of the limited label licenses. Recombinational cloning using gateway and infusion cloning. For traditional restriction enzyme digestion and ligasemediated cloning, use one of the other. Multisite gateway three fragment vector construction kit. An integrated protocol for cloning genes of interest from pcr to agrobacterium transformants via the gateway system was developed.
Onetube protocol for cloning attbpcr products directly into destination vectors. Feb 19, 2019 1918 influenza pandemic survivor interview. The consideration paid for this product grants a limited license with a paid up royalty to use the product pursuant to the. Inserts are usually pcr amplified and vectors are made linear either. Gateway cloning protocols thermo fisher scientific in. Infusion advantage pcr cloning kit user manual protocol no. The fixed cells can also be subjected to immunostaining prior to facs. The consideration paid for this product grants a limited license with a paid up royalty to. The adaptability of the gateway cloning system is ideally suited for students to design and create different types of expression constructs to achieve a particular experimental goal e. Recombination reactions of the gateway cloning system the gateway lr cloning reaction the gateway bp cloni slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Correct design of attb primers for amplification, cloning and expression of a gene in gateway requires consideration of the proper placement of protein expression elements ribosome recognition sequences, start codon, stop codons, reading frame considerations etc. Similar as in gateway cloning, any t1 r competent cells with a transformation efficiency of greater than 1. Recombinational cloning using gateway and infusion. The gateway technology is a universal cloning method based on.
Gibson assembly employs three enzymatic activities in a singletube reaction. This product is covered by limited label licenses see section 1. Converting your favorite set of cloning vectors to gateway technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression. Streamline cloning of genes into binary vectors in. Gateway cloning is an alternative or complementary cloning method to traditional digestligation and gibson assembly that relies on proprietary recombinases from thermofisher. It makes use of the 3 5activity of t4 dna polymerase to create very specific 1015 base single overhangs in the expression vector. Mix 12 ml of bp reaction mix with 50 ml competent dh5a cells for the preparation see the appropriate protocol. A gateway cloning vector set for highthroughput functional analysis of genes in plantaw mark d. Pdf dissociation for fluorescence activated cell sorting facs. Gateway cloning is a multistep recombinational cloning method which eliminates the need for classical restriction enzyme and ligase real cloning for the transfer of genes between vec. The integration process lysogeny is catalyzed by 2 enzymes. Gateway cloning gives researchers the opportunity to easily transfer dna fragments into. Onetube protocol for cloning attbpcr products directly into.
Pcr cloning system with gateway technology order number. Cloning methods gateway creating a gateway entry clone. The infusion cloning products allow ligationindependent cloning of pcr products into any vector, at any site of linearization. This details the methods we used when it was necessary to clone those pcr products which gave mixed or ambiguous sequence data, with the aim. Cloning technologies for protein expression and purification. Molecular cloning is a basic technique used in a molecular biology labs. Invitrogen gateway cloning technology has been cited by life science researchers more than 2,000 times. Protein extraction and purification protein purification tags, histidine and gst tags, imac duration. Here we focus on the 1fragment cloning approach, but we provide tips to help you move onto multisite gateway cloning easily. The use of pcr fragments as building blocks for gateway cloning approaches has been described, however, to our knowledge this has not previously been adapted to freshly ligated grna. We are using pcr products from the above method, cleaned up through an isopropanol precipitation step, as inserts for cloning into the pstblue1 vector. For more information about the gateway technology, see the next page. Invitrogen market a number of kits and cloning vectors designed to simplify the cloning and subcloning of dna fragments such as those generated by pcr amplification.
Mix 1 ml of bp reaction mix with 50 ml competent dh5a cells for the preparation see the appropriate. Briefly centrifuge the pgem t or pgem t easy vector and control insert dna tubes to collect contents at the bottom of the tube. The gateway cloning system, invented and commercialized by invitrogen life technologies since the late 1990s. Invitrogen gateway recombination cloning technology circumvents traditional restriction enzyme based cloning limitations, enabling you to access virtually any expression system in just a few simple steps. Pdf maris preparation of fixed dissociated cells for facs and subsequent rnaseq yes, this means the cells are fixed immediately after dissociation and before facs. Converting your favorite set of cloning vectors to gateway technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression process. The gateway cloning system, invented and commercialized by invitrogen since 2000 hartley et. Gateway technology with clonase ii thermo fisher scientific. The gateway cloning technology is based on the sitespecific recombination system used by phage l to integrate its dna in the e. It has effectively replaced the use of restriction endonucleases and ligases. Quick protocol 1 pgemt and pgemt easy vector systems instructions for use of products a60, a80, a3600 and a3610.
With this you can perform both standard 1 fragment and multsite gateway cloning. By use of this product, you accept the terms and conditions of the limited. Cloning methods gateway one tube protocol to create a. Follow the protocol for the transformation of plasmid dna to chemically competent e. The infusion cloning reaction, which takes as little as 15 minutes, is specific and. Infusion advantage protocol overview the table below is a general outline of the protocol used in the infusion advantage pcr cloning kits. This product is covered by limited label licenses see section1.
To express your gene of interest using the gatewayz technology. Invitrogen gateway technology life technologies mafiadoc. Gateway cloning protocols thermo fisher scientific us. We developed onestep sequence and ligationindependent cloning slic as a simple, costeffective, timesaving, and versatile cloning method. To enable efficient gateway cloning, the reverse primer must contain the following structure. In this manual, we include a protocol for isolating the luciferase gene from dna using restriction digestion. Important the multisite gateway threefragment vector construction kit is. This product is covered by a limited label license see section 1. The gateway cloning system, invented and commercialized by invitrogen since the late 1990s, is a molecular biology method that enables researchers to efficiently transfer dnafragments between. For rapid topo a variety of gateway entry vectors are available from invitrogen to facilitate cloning of pcr products, we recommend using the pentrdtopo or pentrsddtopo cloning kits. This vector is available as part of a kit from novagen catalogue number 701914 which contains everything needed for the ligations and transformations, including the novablue.
Ligase independent cloning lic ligase independent cloning lic is a simple, fast and relatively cheap method to produce expression constructs. Multisite gateway three fragment vector construction. Gateway pdonr vectors hebrew university of jerusalem. I normally make the complementary part of my primers 18 bp long and make sure that there are enough bases behind a restriction site neb 249 so you can do a. Gateway cloning protocols thermo fisher scientific fr. Gateway cloning is a universal cloning technique developed by invitrogen life technologies. From protein expression to functional analysis, gateway cloning technology is applicable for a variety of research areas, for truly. Amongst the most popular are the gateway and topo kits. Cloning methods recombination cloning systems gateway.
Particularly for gateway, hundreds of vectors initially constructed for use with real have been made gatewaycompatible, not only for protein expression and purification but also for many in vivo. Ligation independent cloning lic is a technique developed in the early 1990s as an alternative to restriction enzymeligase cloning. It is a molecular biology method that enables researchers to efficiently transfer dna fragments between plasmids using an appropriate set of recombination sequences, the gateway att sites, and two proprietary enzyme mixes, called lr clonase, and bp clonase. Curtis and ueli grossniklaus institute of plant biology and zu. Bp cloning primer design for pcr sequences to facilitate gateway cloning sequences required for efficient expression of the native protein i.
128 1382 1265 627 643 788 1116 1667 1582 1031 1061 570 754 413 442 1235 742 853 976 309 1643 35 237 188 130 1386 489 611 316 132 988 1352 343 995 680