The neb pcr cloning kit contains a sufficient supply of materials to perform 20 x 10. An alternative to cloning, called the polymerase chain reaction pcr, can be used to directly amplify rare specific dna sequences in a complex mixture when the ends of the sequence are known. Qiagen pcr cloningplus kit transformation protocol. My problem i cant seem to get a 2kb pcr amplicon cloned into the kit s entry vector this is supposed to occur during the 5 minute topo cloning reaction. The pdrive cloning vector contains the standard sequencing annealing sites for m forward and reverse, as well as t7 and sp6 primers.
Topo ta cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of taq polymeraseamplified pcr products into a plasmid vector. Creator cloning kits the creator pdnr cloning kit cat. Instaclone pcr cloning kit 30 rxns k1214 from thermo fisher. Almost everyone has heard of dolly, the cloned sheep born in 1996 but what about the. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Strataclone pcr cloning kit 5 solopack pcr cloning competent cells the provided solopack pcr cloning competent cells express cre recombinase, in order to circularize the linear dna molecules produced by topoisomerase imediated ligation. The strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products at room temperature, using the efficient dna rejoining activity of dna topoisomerase i and the dna. The pcr script cloning kits are convenient for cloning pcr fragments generated with any pcr enzyme. Protocols for cloning without restriction enzymes or.
In the cloning process, the dna is removed from cells, manipulations of the dna are carried out in a testtube, and the dna is subsequently put back into cells. Assemble ligation reactions using the chart below as a guide. Therefore, it takes only one hour from the completion of pcr to cell plating. Kit for sequencing fiveminute cloning of taq polymeraseamplified pcr products for sequencing catalog nos. All components of the exactstart fulllength cdna library cloning kit are free of. Strataclone vector mix, strataclone cloning buffer, strataclone control insert 5 ngl, solopack pcr cloning competent cells, puc18 control plasmid 0. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or. The donor vector in this kit, pdnr1r is designed to increase recovery of. My problem i cant seem to get a 2kb pcr amplicon cloned into the kits entry. Tim sanchez julie robinson mary buchanan stratagene.
The exactstart fulllength cdna library cloning kit is functiontested in a control reaction. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. In the cloning process, the dna is removed from cells, manipulations. Instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs generated by taq dna polymerase and other thermostable dna polymerases, which lack proofreading activity. What sequencing primers can i use with the pdrive cloning. Enhanced pcr cloning system pcrscript cloning kits. Strataclone pcr cloning kit 5 map for the strataclone pcr cloning vector pscaampkan feature nucleotide position. Genscripts proprietary cloneez seamless cloning technology is a cuttingedge, recombinationbased cloning technology that can accurately and efficiently assemble genes and gene fragments in any combination of sequences.
Fiveminute cloning of taq polymeraseamplified pcr products. Instaclone pcr cloning kit 30 rxns k1214 from thermo. Protocol for exactstart fulllength cdna library cloning kit. Oct 24, 20 hello everyone, my first post on protocolonline. Strataclone pcr cloning kit from agilent technologies. 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. These kits are available for both bluntend and ua cloning. Kit for sequencing version j 052 250276 topo ta cloning. 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. Any other blunt or stickyend dna fragment can be cloned. The clonejet pcr cloning kit is an advanced positive selection system. Sequence and ligase independent cloning slic as can be deduced from the name, ligase is not needed in the slic method.
Map for the strataclone pcr cloning vector pscaampkan. The strataclone pcr cloning kit allows highefficiency, 5minute cloning of pcr products at room temperature, using the efficient dna rejoining activity of dna topoisomerase i and the dna recombination activity of cre recombinase. Thermo scientific clonejet pcr cloning kit, also available with dh10b cells k123120, is an advanced positive selection system for highefficiency cloning of. The cells are provided in a convenient singletube transformation format. The dna insert can be readily excised from the versatile polylinker of ptz57rt, sequenced using standard mpuc primers or in vitro transcribed with t7 rna. Taq dna polymerase catalyzes the nontemplate directed addition of an adenine residue to the 3. Description the genscript cloneez pcr cloning kit is designed for the quick pcr cloning, it is especially powerful in highthroughput cloning of pcr products into any destination vector effectively by sidestepping the tedious and limiting tasks such as selecting proper restriction enzymes, phosphatases, or ligases. An introduction to pcr cloning pcr products generated using a nonproofreading dna polymerase which lacks 3 to 5 exonuclease activity, such as taq dna polymerase, are left with a single templateindependent nucleotide, deoxyadenosine a, at the 3.
Msds documents are not included with product shipments. The kit must produce 107 colonies from 250 ng of the exactstart rna control template when using. Ive got a problem with invitrogens pentr d topo cloning kit. K4575j10, k457501, k457540, k458001, k458040, k459501, k459540 a limited label license covers this product see purchaser notification. The cloning vector was prepared by cutting nzytech. The pcrscript cloning kits are convenient for cloning pcr fragments generated with any pcr. The cornerstone of infusion cloning technology is clontechs proprietary infusion enzyme, which fuses. Clonexpress ii one step cloning kit,rapid cloning,vazyme. Tvector cloning, or ta cloning, is a convenient method for cloning pcr products generated with taq dna polymerase. A cloning vector is a small piece of dna that can be stably maintained in an organism, and into which a foreign dna fragment can be inserted for cloning purposes. The kit must produce 107 colonies from 250 ng of the exactstart rna control template when using epicentres transformax ec100 electrocompetent e.
Design of primers for pcr reaction for quadplex grna cloning the scalability of the multiplex grna cloning kit allows for. Cloning using the invitrogen ta cloning kit whats needed. The addition of a a by taq dna polymerase is not required and has no effect on cloning efficiency. The kit also allows direct cloning from amplification reactions without purification, and works well whether or not the primers used in the pcr possess 5. Lastly, if your pcr product is longer than usual i. Description high efficiency pcr cloning, features dna topoisomerase i and cre recombinase technologies be the first to write a. Limited label license for strataclone pcr cloning kits. No ligase, post pcr procedures, or pcr primers containing specific sequences are required. Thermo scientific clonejet pcr cloning kit for 20 reactions. Our transformation protocol is often faster than the transformation of commercially available competent cells. Primers are also provided, allowing screening for inserts by colony pcr andor sequencing. The cloning vector may be dna taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The mighty tacloning kit allows for fast, easy cloning of pcr products with this ta cloning method. Protocols for cloning without restriction enzymes or ligases.
For descriptions of symbols on product labels or product documents, go to. Transformation protocol for neb pcr cloning kit the following protocol is designed for neb 10beta competent e. Instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs generated by taq dna polymerase and other thermostable dna polymerases, which. The high quality readytouse ta cloning vector ptz57rt was prepared by linearizing the ptz57r plasmid with eco32i and tailing with. Cloning of taq polymeraseamplified pcr products thermo. The clonejet pcr cloning kit is an advanced positive selection system for the highest efficiency cloning of pcr products generated with.
Ligationfree cloning highthroughput cloning seamless dna fusion multiple pcr fragment assembly uracil excisionbased cloning artificial microrna homologous recombination gateway. Nzya pcr cloning kit was designed to allow the direct cloning of pcr products with 5. The high quality ta cloning vector ptz57rt is ready to use for efficient ligation with pcr products providing high cloning yields and low background. Thermo scientific clonejet pcr cloning kit, also available with dh10b cells, is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna. Map for the strataclone blunt pcr cloning vector pscbampkan 6. A overhangs, which result from amplifications using nonproofreading dna polymerases such as nzytaq dna polymerase. The pcr fragments can be generated by taq dna polymerase or other high fidelity dna polymerases. The strataclone pcr cloning kit was used to clone pcr fragments from a common fluorescent protein 664 bp. Nov 08, 2018 lastly, if your pcr product is longer than usual i. Dna cloning cloning is the process of moving a gene from the chromosome it occurs in naturally to an autonomously replicating vector. Strataclone pcr cloning kit 7 using plasmid dna as pcr template genomic dna, plasmid dna, or cdna may be used as template for pcr amplification prior to cloning. Cannot clone 2kb insert into pentr d topo cloning vector. Limited label license for strataclone ultra blunt pcr cloning kits. Figure 1 in the qiagen pcr cloning kit handbook shows the pdrive.
Clonejet pcr cloning kit and are available as standalone strains or. Cloning technology was invented during the twentieth century and now is poised to help define the twentyfirst. Clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase. Note do not use taq dna polymerase, or enzyme blends containing predominantly taq dna polymerase, to amplify fragments for cloning using the strataclone blunt pcr cloning kit. Transformation protocol for neb pcr cloning kit neb. The thermo scientific instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs 1. The strataclone pcr cloning kit was used to clone pcr fragments from a common fluorescent protein 664 bp, gammaglobin 1.