Invitrogen zero blunt topo pcr cloning kit, with pcrblunt iitopo vector, one shot top10 chemically competent e. I constructed three plasmid vectors for pcr cloning in this study fig. Generate target dna fragments by shearing, restriction digestion, or pcr. No ligase, post pcr procedures, or pcr primers containing specific sequences are required. Note whether or not the primers are phosphorylated at the 5end. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt. Linearize the vector by restriction enzyme digestion or inverse pcr and purify.
The sureclone ligation kit from amersham pharmacia biotech is intended for cloning pcr products directly into any vector. I use pcrblunt vector and zero blunt pcr cloning kit. Ecorv and smai are the most popular endonucleases utilized to digest vector and produce bluntend. B p u c o r i zeoci n nsi i hind iii asp 718i kpn i ecl 6ii sac i bam hi spe i eco ri eco pst i eco rv not xho nsi i xba dra ii apa i pcrblunt iitopo. 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. Bluntended pcr products, produced by proofreading pcr enzymes, are efficiently ligated to these. Relatively simple construct design and cloning procedure are advantages of blunt end cloning. Pcr cloning vector mix, strataclone pcr cloning kits agilent. Sequence or clone directly into an expression vector. Zero blunt topo pcr cloning provides a highly efficient, 5minute, onestep cloning strategy topo cloning for the direct insertion of blunt end pcr products into a plasmid vector. No ligase, postpcr procedures, or pcr primers containing specific sequences are. Here, we reported a method for generating linearized bluntend vector pbskblunt by pcr. The pcrzerot vector is compatible for both ta cloning and bluntend cloning of pcr products. Increase the speed and efficiency of your pcr product cloning today with the agilent strataclone pcr cloning kit.
When bluntend pcr products are directly cloned into nondigested vectors, smai for pcrzerot or ecorv for pcrzero can be selected, depending on. Pcr products are first subjected to a 30 minute bluntingkinasing reaction to remove overhangs from the pcr products and to phosphorylate the ends, followed. Bluntend vectors generated by polymerase chain reaction. When pcr was in its infancy, researchers found that subcloning pcr products by simple bluntended ligation into bluntended plasmid cloning vectors was not easy. Pdf pxst, a novel vector for ta cloning and bluntend. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Available for both blunt and ua pcr cloning, the kits employ the rejoining activity of dna topoisomerase i with the dna recombination activity of cre recombinase. Ma004 clonesmart cloning kitspsmart vectors lucigen. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. The ends of the vector should not be able to religate because either they are incompatible e. This a overhang can create difficulties when cloning the fragment is your end goal. Ta cloning also known as rapid cloning or t cloning is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. Since the linearized vector and dna fragment are both bluntend in the t4 ligation reaction mixture, it causes the problem of vector selfligation, reducing the possibility of. Left plate serves as the control, with vector backbone only, right plate contains pcr.
It works perfectly, you can reduce lidation volume to 5 ul and use any regular t4 ligase if u run out of the one that is supplied with kit. Dna amplicons by the polymerase chain reaction pcr, require to be introduced. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. Introduction product overview description of the system the zero blunt pcr cloning kit is designed to clone blunt pcr fragments or any blunt dna fragment with a low background of nonrecombinants. Zero blunt pcr cloning kit thermo fisher scientific. Topo cloning vectors for optimized sequencing molecular info. Zero blunt topo pcr cloning kit, with pcrblunt iitopo. Zero blunt topo pcr cloning kit options the zero blunt topo pcr cloning kit for direct insertion of bluntended pcr products into a plasmid vector can be purchased with a variety of competent cells that deliver different advantages depending upon your needs general cloning. The plasmid vector pcrblunt iitopo is supplied linearized with vaccinia virus dna topoisomerase i covalently bound to the 3. Bluntended pcr products, produced by proofreading pcr enzymes, are efficiently ligated to these vector arms in a 5minute ligation reaction by topoisomerase i. Strataclone dna topoisomerase i technology with simple primer design, no. Receive the latest news, hot plasmids, discounts and more.
As a result, only bacterial cells with recombinant plasmids are able to form colonies. Briefly, an inverse pcr was performed with the target vector as a template with a pair of phosphorylated primers, then the pcr products were subject to dpni treatment, agarose gel purification and further, the terminal deoxynucleotidyl transferase was applied to add a t to the 3. 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. Consortium plasmids pcr blunt iitopo pcr blunt iitopo parental vector for topo cloning of blunt pcr products. When the cycle is repeated several times, the net result is a rapid increase in the total number of copies of the target dna. Zero blunt topo pcr cloning kit, without competent cells. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. Zero blunt topo pcr cloning kit thermo fisher scientific. Ligation of a blunt pcr fragment disrupts expression.
If you plan to clone a bluntended pcr fragment generated with a polymerase such as pfu into a phosphatasetreated vector, ensure that your pcr primers have 5. How zero blunt pcr cloning works the zero blunt pcr cloning kit is designed to clone blunt pcr fragments or any blunt dna fragment with a low background of nonrecombinants. This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. See the pcr protocols page for general insert amplification with vent.
Bluntend vectors can be generated by pcr or enzymes digestion. Dna by pcr, cleaved to create ecorv ends and inserted into ecorv cleaved, dephosphorylated litmus 28 vector. However, another thermostable dna polymerase, like taq dna polymerase, adds a single nucleotide base to the 3 end of the dna fragment, usually an adenine, creating an a overhang. Moreover, 5 dephosporylation of the vector is usually required to avoid vector selfligation.
Topo pcr cloning solutions thermo fisher scientific. Pcr product, cloning, tvector, bluntend ligation, high efficiency background amplification and cloning of genes are fundamental techniques in the field of molecular biology. Clonejet pcr cloning kit taq thermo fisher scientific. The atailing protocol and subsequent ligation of two different pcrgenerated fragments were tested for this report. Digital collection of empty plasmid backbones from publications and commercially available sources. Using pcrzero and pcrzerot and applying the golden gate reaction, i developed a direct pcr cloning protocol with nondigested circular. Each cycle involves three steps, which are described in detail above. Tips for restriction cloning at the bench snapgene. With a phosphatasetreated vector, perform a control ligation in which the inserted fragment is omitted. In addition to ta cloning, bluntend pcr products with t ended in the primer could be positively linked to the tvector pgpb2e without atailing treatment tb cloning. T overhangs that can be used for cloning pcr products derived from amplification by taq polymerase, and ecorv, which yields bluntends suitable for cloning pcr products generated by pfu dna polymerases.