Pcrbased cloning from plasmids entered by karin holmberg, kevin janes janes lab protocols updated by cheryl borgman 020817 3 excise the band on the lucite gel holder with a clean razor blade. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. The production of exact copies of a particular gene or dna sequence using genetic engineering techniques is called gene cloning. With this method, the vector and insert are pcr amplified separately. In this method, two pairs of pcr primers are designed and are amplified in two different. 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 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. Qiagen pcr cloning handbook university of illinois. Part of the methods in molecular biology book series mimb, volume 192. Page 1 of 7 please read the infusion hd cloning kit user manual before using this protocolataglance. Part i presents basic pcr protocols, emphasizing optimizing conditions for he amplification of dna fragments of several kilobases in length. 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.
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. Common cloning applications and strategies thermo fisher. Pdf molecular cloning using polymerase chain reaction, an. 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. Rtpcr protocol for highfidelity cloning applications.
Cloning of pcr products, we recommend using the pentrdtopo or pentrsddtopo cloning kits. 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. Qiagen pcr cloning kits are developed, designed, and sold for research purposes only. 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.
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. Pcr cloning enables the dna fragment of interest and the vector to be amplified by pcr and ligated together, without restriction enzyme digestion. Overlap extension pcr cloning article pdf available in methods in molecular biology clifton, n. 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. Gene cloning requirements, principle, steps, applications. 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. Infusion hd multipleinsert cloning protocolataglance. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Left plate serves as the control, with vector backbone only, right plate contains pcr. Infusion advantage pcr cloning kit user manual protocol no. Using these two kits in conjunction helps ensure that accurate amplicons are generated for cloning. The kit uses the linearized and topoisomerase 1 activated pcrxl2topo vector, which is compatible with the cloning of bluntend pcr fragments. Pcr cloning is a method in which doublestranded dna fragments amplified by pcr are ligated directly into a vector. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. Design primers to amplify fragments andor vector with appropriate overlaps see pages 410. Kits with competent cells are available with one shot chemically or electrocomp competent cells as described in the following table see page. Pcr amplify fragments using a highfidelitydna polymerase. For traditional restriction enzyme digestion and ligasemediated cloning, use one of the other pentr vectors.
Add 2 l of cloning enhancer to 5 l of each pcr reaction insert or linearized 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. Oct 12, 2011 although a variety of methods and expensive kits are available, molecular cloning can be a timeconsuming and frustrating process. 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. For cloning bluntend pcr products generated by proofreading dna polymerases, such as pfu dna polymerase. 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. For more information about the features of the entry vectors, see our web site. Use a thermal cycler to incubate at 37c for 15 min, then at 80c for 15 min. The term gene cloning, dna cloning, molecular cloning, and recombinant dna technology all refer to same technique.
Infusion advantage protocol overview the table below is a general outline of the protocol used in the infusion advantage pcr cloning kits. Print bookmark share for qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format file size language download get adobe reader. Prepare linearized vector by pcr amplificationusing a highfidelitydna polymerase or by restriction digestion. Molecular cloning using polymerase chain reaction, an. Learn more about standard pcr, including what it is, on our pcr basics page. Cloning protocols bluntend cloning protocol for cloning bluntend pcr products generated by proofreading dna polymerases, such as pfu dna polymerase. Pcr cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than. 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. Infusion hd multipleinsert cloning protocolataglance 121416 takara bio usa, inc. The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. If the dna end structure of the pcr products is not specified by the supplier of the dna polymerase, follow the stickyend cloning protocol. For qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format. As a result, only bacterial cells with recombinant plasmids are able to form colonies. 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 pgemt and pgemt easy vector systems are designed for just that purpose. When cloning by pcr, it is especially important to run the product on a gel. After plating, i randomly selected 5 colonies from. Protocol for cloning insert into as2 series plasmids sticky end pcr method. The qiagen pcr cloning kit and box i of the qiagen pcr cloningplus kit containing ligation master mix and pdrive cloning vector should be stored at 20c in a constanttemperature freezer or at 70c immediately upon receipt. 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. Infusion cloning procedure w cloning enhancer treatment 1. 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. The most basic need in pcr subcloning is a simple, general cloning vector.
The invitrogen topo xl2 complete pcr cloning kit provides all the necessary elements for highly efficient cloning of extralong pcr products up to kb. 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. This is a basic pcr protocol using taq dna polymerase. Pcrbased cloning from plasmids entered by karin holmberg. Design of the primers for overlap extension pcr cloning. 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. Part of the methods in molecular biology book series mimb, volume 67. Sticky end pcr cloning zeng, 1998 that allows one to generate sticky end by using standard pcr method is described below. Cloning shrna libraries short loop, perfect complementarity, into a modified psicor vector under the normal u6 promoter. 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. I normally make the complementary part of my primers 18 bp long and make sure that there are enough bases behind a. Molecular cloning is a basic technique used in a molecular biology labs.
A pcr cloning strategy called sticky end pcr cloning. A wide variety of protocols from addgene that can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations. If you used more than 100 ng of dna template, extend the 37c incubation to 20 min. 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. Cloning protocolstips by astrid california institute of. Library cloning protocol shrna context mcmanus lab. Overview of strataclone pcr cloning technology using the method summarized in figure 1, strataclone pcr cloning. Prepare a reaction mixture for the appropriate number of samples to be amplified. First, pcr primers are designed that share 15 bases of homology with the sequence at the ends of the linearized cloning vector i.
For my cloning experiment, i first ligated my insert 1. Protocol for cloning insert into as2 series plasmids sticky end pcr method introduction. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying pcr in gene cloning. Find additional protocols for other polymerases or advanced pcr techniques in the protocols section of our pcr technologies guide. 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. Protocol for cloning the luciferase gene into the pgem vector. Jan 04, 2020 gene cloning requirements, principle, steps, applications.