Qiagen pcr cloning handbook university of illinois. For my cloning experiment, i first ligated my insert 1. Left plate serves as the control, with vector backbone only, right plate contains pcr. Pcr cloning offers some advantages over traditional cloning which relies on digesting doublestranded dna inserts with restriction enzymes to create compatible ends, purifying and isolating sufficient amounts, and ligating into a similarly treated vector of choice see insert. Prepare linearized vector by pcr amplificationusing a highfidelitydna polymerase or by restriction digestion. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols.
This pcr cloning kit contains an optimized cloning mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to. Prepare a reaction mixture for the appropriate number of samples to be amplified. Background in its simplest form, pcr based cloning is about making a copy of a piece of dna and at the same time adding restriction sites to the ends of that piece of dna so that it can be easily cloned into a plasmid of interest. In this method, two pairs of pcr primers are designed and are amplified in two different. Oct 12, 2011 although a variety of methods and expensive kits are available, molecular cloning can be a timeconsuming and frustrating process. Page 1 of 7 please read the infusion hd cloning kit user manual before using this protocolataglance. Part of the methods in molecular biology book series mimb, volume 67. In addition to gel purification, the following minor protocol modifications can facilitate the recovery of clones containing long 3 kb pcr product inserts. Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate.
Pcr cloning is a method in which doublestranded dna fragments amplified by pcr are ligated directly into a vector. Protocol for cloning insert into as2 series plasmids sticky end pcr method introduction. From molecular cloning to genetic engineer ing is divided into seven parts, each containing a collection of chapters address ing a general approach or goal. Rtpcr protocol for highfidelity cloning applications. Pdf molecular cloning using polymerase chain reaction, an. Library cloning protocol shrna context mcmanus lab. Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than. Here the researcher will find readily reproducible methods for all the major aspects of pcr use, including pcr optimization, computer programs for pcr primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on longdistance pcr and gcrich template amplification. Here we report a highly simplified, reliable, and efficient pcr based cloning technique to insert any dna fragment into a plasmid vector or into a gene cdna in a vector at any desired position. Infusion advantage protocol overview the table below is a general outline of the protocol used in the infusion advantage pcr cloning kits. Cloning of pcr products, we recommend using the pentrdtopo or pentrsddtopo cloning kits. Pcr based cloning is incredibly versatile and allows for nearly any piece of dna to be placed into a backbone vector of choice with minimal limitations.
The kit uses the linearized and topoisomerase 1 activated pcrxl2topo vector, which is compatible with the cloning of bluntend pcr fragments. A pcr cloning strategy called sticky end pcr cloning. Part of the methods in molecular biology book series mimb, volume 192. Gene cloning requirements, principle, steps, applications. Qiagen pcr cloning kits are developed, designed, and sold for research purposes only. 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. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3.
Jan 19, 2015 due to the emergence of novel and efficient pcr reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of pcr cloning starting from the primer design, performing pcr, sequencing pcr products, analysis of the sequencing data, and finally the assessment of gene expression. Sticky end pcr cloning zeng, 1998 that allows one to generate sticky end by using standard pcr method is described below. Pcrbased cloning from plasmids entered by karin holmberg. Learn more about standard pcr, including what it is, on our pcr basics page. The production of exact copies of a particular gene or dna sequence using genetic engineering techniques is called gene cloning. The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. Design of the primers for overlap extension pcr cloning. Cloning protocolstips by astrid california institute of.
For more information about the features of the entry vectors, see our web site. Infusion hd multipleinsert cloning protocolataglance 121416 takara bio usa, inc. With this method, the vector and insert are pcr amplified separately. Infusion cloning procedure w cloning enhancer treatment 1.
Infusion advantage pcr cloning kit user manual protocol no. The most basic need in pcr subcloning is a simple, general cloning vector. The strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products, using the efficient dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase. Part i presents basic pcr protocols, emphasizing optimizing conditions for he amplification of dna fragments of several kilobases in length. Overlap extension pcr cloning article pdf available in methods in molecular biology clifton, n. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying pcr in gene cloning. 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 of then sequencing some of the resultant clones to see if we had pcred homeologous sequences with our initial pcr reaction.
Use a thermal cycler to incubate at 37c for 15 min, then at 80c for 15 min. For traditional restriction enzyme digestion and ligasemediated cloning, use one of the other pentr vectors. Kits with competent cells are available with one shot chemically or electrocomp competent cells as described in the following table see page. Overview of strataclone pcr cloning technology using the method summarized in figure 1, strataclone pcr cloning. This is a basic pcr protocol using taq dna polymerase. The vectors provide convenient t7 and sp6 promoters that can serve as binding sites for sequencing primers, or for promoting in vitro transcription on either strand of the insert when the. If the dna end structure of the pcr products is not specified by the supplier of the dna polymerase, follow the stickyend cloning protocol. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a dna insert into the cloning site. This page seeks to provide an introduction to cloning techniques and methodologies for the beginning researcher and assumes ready knowledge in molecular biology. The invitrogen topo xl2 complete pcr cloning kit provides all the necessary elements for highly efficient cloning of extralong pcr products up to kb. Add 2 l of cloning enhancer to 5 l of each pcr reaction insert or linearized vector. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1.
Design primers to amplify fragments andor vector with appropriate overlaps see pages 410. A wide variety of protocols from addgene that can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations. This allows you to visualize that your pcr product is the anticipated size and that your band is strong indicating that the pcr reaction worked and that you have a sufficient amount of dna. If you used more than 100 ng of dna template, extend the 37c incubation to 20 min. After plating, i randomly selected 5 colonies from.
When cloning by pcr, it is especially important to run the product on a gel. First, pcr primers are designed that share 15 bases of homology with the sequence at the ends of the linearized cloning vector i. For cloning bluntend pcr products generated by proofreading dna polymerases, such as pfu dna polymerase. Cloning protocols bluntend cloning protocol for cloning bluntend pcr products generated by proofreading dna polymerases, such as pfu dna polymerase. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. Cloning shrna libraries short loop, perfect complementarity, into a modified psicor vector under the normal u6 promoter. In this manual, we include a protocol for isolating the luciferase gene from dna using restriction digestion and cloning it into the multiple cloning region of a vector. Using these two kits in conjunction helps ensure that accurate amplicons are generated for cloning. The protocol below details how to use these kits for rt pcr when cloning and offers a highlevel overview of steps integral to the entire cloning process.
Print bookmark share for qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format file size language download get adobe reader. The pgemt and pgemt easy vector systems are designed for just that purpose. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Arguably one of the most powerful laboratory techniques ever discovered, pcr combines the unique attributes of being very sensitive and specific with a great degree of flexibility. They are not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the food and drug administration in the usa or the appropriate regulatory authorities in the country of use. Find additional protocols for other polymerases or advanced pcr techniques in the protocols section of our pcr technologies guide.
Pcr cloning enables the dna fragment of interest and the vector to be amplified by pcr and ligated together, without restriction enzyme digestion. Jan 04, 2020 gene cloning requirements, principle, steps, applications. Protocol for cloning the luciferase gene into the pgem vector. 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. Pcr amplify fragments using a highfidelitydna polymerase. Pcr cloning differs from traditional cloning in that the dna fragment of interest, and even the vector, can be amplified by the polymerase chain reaction pcr and ligated together, without the use of restriction enzymes. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Infusion hd multipleinsert cloning protocolataglance. For qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format. Common cloning applications and strategies thermo fisher. Molecular cloning is a basic technique used in a molecular biology labs. The term gene cloning, dna cloning, molecular cloning, and recombinant dna technology all refer to same technique. Protocol for cloning insert into as2 series plasmids sticky end pcr method.
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