If necessary, repair the dna ends to make them blunt, with 5 phosphate groups. 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. When bluntend pcr products are directly cloned into nondigested vectors, smai for pcrzerot or ecorv for pcrzero can be selected, depending on. Consortium plasmids pcr blunt iitopo pcr blunt iitopo parental vector for topo cloning of blunt pcr products. 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. The ends of the vector should not be able to religate because either they are incompatible e. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt iitopo vector, one shot top10 chemically competent e. 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. 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. 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. See the pcr protocols page for general insert amplification with vent. Digital collection of empty plasmid backbones from publications and commercially available sources. Zero blunt topo pcr cloning kit, without competent cells.
Clonejet pcr cloning kit taq thermo fisher scientific. For vector construction, a minimal polylinker was designed figure 1 a with the inclusion of restriction sites for xcmi, which produce 3. Topo pcr cloning solutions thermo fisher scientific. A novel series of highefficiency vectors for ta cloning and bluntend. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. 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. No ligase, postpcr procedures, or pcr primers containing specific sequences are. Pcr product, cloning, tvector, bluntend ligation, high efficiency background amplification and cloning of genes are fundamental techniques in the field of molecular biology. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. When pcr was in its infancy, researchers found that subcloning pcr products by simple bluntended ligation into bluntended plasmid cloning vectors was not easy. Moreover, 5 dephosporylation of the vector is usually required to avoid vector selfligation.
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. Tips for restriction cloning at the bench snapgene. The sureclone ligation kit from amersham pharmacia biotech is intended for cloning pcr products directly into any vector. When the cycle is repeated several times, the net result is a rapid increase in the total number of copies of the target dna. Bluntend vectors generated by polymerase chain reaction. Dna amplicons by the polymerase chain reaction pcr, require to be introduced. Zero blunt topo pcr cloning kit user guide thermo fisher. Bluntended pcr products, produced by proofreading pcr enzymes, are efficiently ligated to these.
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. The pcrzerot vector is compatible for both ta cloning and bluntend cloning of pcr products. This a overhang can create difficulties when cloning the fragment is your end goal. The atailing protocol and subsequent ligation of two different pcrgenerated fragments were tested for this report. Ecorv and smai are the most popular endonucleases utilized to digest vector and produce bluntend.
Sequence or clone directly into an expression vector. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a dna insert into the cloning site. Strataclone dna topoisomerase i technology with simple primer design, no. Zero blunt pcr cloning kit thermo fisher scientific. 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. Pcr products are first subjected to a 30 minute bluntingkinasing reaction to remove overhangs from the pcr products and to phosphorylate the ends, followed. With a phosphatasetreated vector, perform a control ligation in which the inserted fragment is omitted.
Zero blunt topo pcr cloning kit thermo fisher scientific. The strataclone blunt pcr cloning vector mix contains two bluntended dna arms, each charged with topoisomerase i on one end and containing a loxp recognition sequence on the other end. 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. Invitrogens zeroblunt vector and quantum biotechnologies quantox carry a. Ma004 clonesmart cloning kitspsmart vectors lucigen.
Generate target dna fragments by shearing, restriction digestion, or pcr. 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. Bluntend vectors can be generated by pcr or enzymes digestion. 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 vector mix, strataclone pcr cloning kits agilent. Zero blunt topo pcr cloning kit, with pcrblunt iitopo. Ligation of a blunt pcr fragment disrupts expression. Receive the latest news, hot plasmids, discounts and more.
Using pcrzero and pcrzerot and applying the golden gate reaction, i developed a direct pcr cloning protocol with nondigested circular. A novel series of highefficiency vectors for ta cloning. As a result, only bacterial cells with recombinant plasmids are able to form colonies. 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. Topo cloning vectors for optimized sequencing molecular info. I constructed three plasmid vectors for pcr cloning in this study fig. The plasmid vector pcrblunt iitopo is supplied linearized with vaccinia virus dna topoisomerase i covalently bound to the 3. 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. 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. No ligase, post pcr procedures, or pcr primers containing specific sequences are required. Cloning for the direct insertion of bluntend pcr products into a plasmid vector. Pdf pxst, a novel vector for ta cloning and bluntend. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt. Linearized, blunt end vector and blunt end insert are then combined and incubated with t4 dna ligase in appropriate buffer in order to form phosphodiester bonds between insert and vector.
Left plate serves as the control, with vector backbone only, right plate contains pcr. Each cycle involves three steps, which are described in detail above. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. The technique relies on the ability of adenine a and thymine t complementary basepairs on different dna fragments to hybridize and, in the presence of ligase, become ligated together. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources.