Using pcrzero and pcrzerot and applying the golden gate reaction, i developed a direct pcr cloning protocol with nondigested circular. Ma004 clonesmart cloning kitspsmart vectors lucigen. Consortium plasmids pcr blunt iitopo pcr blunt iitopo parental vector for topo cloning of blunt pcr products. 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. Digital collection of empty plasmid backbones from publications and commercially available sources. When bluntend pcr products are directly cloned into nondigested vectors, smai for pcrzerot or ecorv for pcrzero can be selected, depending on. Here, we reported a method for generating linearized bluntend vector pbskblunt by pcr.
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. Pcr cloning vector mix, strataclone pcr cloning kits agilent. A novel series of highefficiency vectors for ta cloning and bluntend. Pcr amplification with a proofreading polymerase, like pfu dna polymerase, will leave you with a blunt end. When pcr was in its infancy, researchers found that subcloning pcr products by simple bluntended ligation into bluntended plasmid cloning vectors was not easy. Topo pcr cloning solutions thermo fisher scientific. The clonejet pcr cloning kit contains a novel, readytouse positive selection cloning vector pjet1. Strataclone dna topoisomerase i technology with simple primer design, no. 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. 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.
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. Zero blunt topo pcr cloning kit, without competent cells. See the pcr protocols page for general insert amplification with vent. Sequence or clone directly into an expression vector. Zero blunt pcr cloning kit thermo fisher scientific. Invitrogen zero blunt topo pcr cloning kit, with pcrblunt iitopo vector, one shot top10 chemically competent e. Pcr products are first subjected to a 30 minute bluntingkinasing reaction to remove overhangs from the pcr products and to phosphorylate the ends, followed.
Invitrogen zero blunt topo pcr cloning kit, with pcrblunt. Bluntend vectors can be generated by pcr or enzymes digestion. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a dna insert into the cloning site. Note whether or not the primers are phosphorylated at the 5end. With a phosphatasetreated vector, perform a control ligation in which the inserted fragment is omitted. Pdf pxst, a novel vector for ta cloning and bluntend. 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. Invitrogens zeroblunt vector and quantum biotechnologies quantox carry a. I constructed three plasmid vectors for pcr cloning in this study fig. 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.
Bluntended pcr products, produced by proofreading pcr enzymes, are efficiently ligated to these vector arms in a 5minute ligation reaction by topoisomerase i. The sureclone ligation kit from amersham pharmacia biotech is intended for cloning pcr products directly into any vector. Generate target dna fragments by shearing, restriction digestion, or pcr. 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. If necessary, repair the dna ends to make them blunt, with 5 phosphate groups.
Receive the latest news, hot plasmids, discounts and more. No ligase, postpcr procedures, or pcr primers containing specific sequences are. 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. No ligase, post pcr procedures, or pcr primers containing specific sequences are required. Clonejet pcr cloning kit taq thermo fisher scientific. 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. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Pcr product, cloning, tvector, bluntend ligation, high efficiency background amplification and cloning of genes are fundamental techniques in the field of molecular biology. Cloning for the direct insertion of bluntend pcr products into a plasmid vector. 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. Ligation of a blunt pcr fragment disrupts expression. The pcrzerot vector is compatible for both ta cloning and bluntend cloning of pcr products. 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. 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.
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. The plasmid vector pcrblunt iitopo is supplied linearized with vaccinia virus dna topoisomerase i covalently bound to the 3. Each cycle involves three steps, which are described in detail above. Zero blunt topo pcr cloning kit user guide thermo fisher. For vector construction, a minimal polylinker was designed figure 1 a with the inclusion of restriction sites for xcmi, which produce 3. 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.
The ends of the vector should not be able to religate because either they are incompatible e. 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. Topo cloning vectors for optimized sequencing molecular info. This a overhang can create difficulties when cloning the fragment is your end goal.
Dna amplicons by the polymerase chain reaction pcr, require to be introduced. 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. Left plate serves as the control, with vector backbone only, right plate contains pcr. Pcr cloning with lowno background a 500 bp pcr product incubated with the linearized vector in a 3. Tips for restriction cloning at the bench snapgene. When the cycle is repeated several times, the net result is a rapid increase in the total number of copies of the target dna. 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. Bluntended pcr products, produced by proofreading pcr enzymes, are efficiently ligated to these.
I use pcrblunt vector and zero blunt pcr cloning kit. No ligase, postpcr procedures, or pcr primers containing specific sequences are required. Dna by pcr, cleaved to create ecorv ends and inserted into ecorv cleaved, dephosphorylated litmus 28 vector. Moreover, 5 dephosporylation of the vector is usually required to avoid vector selfligation. Zero blunt topo pcr cloning kit thermo fisher scientific. The atailing protocol and subsequent ligation of two different pcrgenerated fragments were tested for this report. Bluntend vectors generated by polymerase chain reaction.