Strataclone pcr cloning kit from stratagene biocompare. Ta cloning kit designed for cloning directly from a pcr reaction. Specific pcr products of pcr reaction mixture without any purification. Can i use the neb pcr cloning kit featuring pminit 2. Download a pdf containing pricing for our full product list. Can the cloning kit be used for inserts that are not necessarily pcr amplicons. The pcr was carried out same way as the nested amplification with the. The gene fragments of ten shrimp species amplified by longer pcr were. The special ligation system provided by the kit enables customs to finish ligation in 12 hour.
Sixty independent clones were analyzed by colony pcr using the smartiiaprimer and the genespecific gldpa race6 oligonucleotide 5. Extended protocol and realtime pcr dataset for shrimp. In the manual, it recommends using linearized vectors with a concentration of. Instaclone pcr cloning kit has been combined with the transformaid bacterial transformation kit a set of solutions for preparation of chemically competent e. Such polymerases attach a single a overhang to their reaction products, which can hybridize to the u overhang of the pdrive cloning vector. Instaclone pcr cloning kit 30 rxns k1214 from thermo. Pcrscript amp cloning kit 3 introduction the stratagene pcrscript amp cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. Infusion advantage pcr cloning kit user manual protocol no. The recombinants were screened by colony pcr with respective primer sets and plasmids from positive clones were purified using a qiaprep spin miniprep kit. How to obtain the entire sequence of ptz57rt for ta cloning. Thermo scientific instaclone pcr cloning kit for 30.
Pcr products were analyzed through agarose gel 1% visualization. Dreamtaq dna polymerase uses the same reaction set up and cycling. Instaclone pcr cloning kit molecular cloning polymerase. Topoisomerase technology topoisomerase technology requires no special primers and no ligases it is as easy as cloning comes.
Measured in efficiency, time consideration and simplicity, this product by far justifies the cost of kit. May 01, 2019 gelanalyze the pcr product for specificity and yield before cloning. Pdf reproducibility of vibrionaceae population structure in. I did ta cloning with instaclone pcr cloning kit and i got some weird results. By using this kit, we can select specific pairs of restriction enzyme sites at both the 5 and 3 ends of the desired gene to clone into a vector of interest. 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 give a low. Instaclone pcr cloning kit 30 rxns from thermo fisher scientific.
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. Dna cloning and sequencing amplicons containing the its2 region and some of the flanking 5. It is ideal for phosphorylated or nonphosphorylated dna fragments. Primer design using in silico created final dna sequence file. It takes only one hour from the completion of pcr to cell plating. Sixty independent clones were analyzed by colony pcr. Protocols for cloning without restriction enzymes or ligases. Race pcr products were cloned into ptz57rt vector by instaclone pcr cloning kit fermentas life sciences. Pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs. Specific pcr products of instaclone pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific. Highly effective and safe polymeric inhibitors of herpes. Instaclone pcr cloning kit 30 rxns k1214 from thermo fisher. Thermo scientific clonejet pcr cloning kit is an advanced positive selection system for highefficiency cloning of pcr products generated with any thermostable dna polymerase.
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. Aug 29, 2008 pcr products were gelpurified with the nucleospin extract ii kit machereynagel gmbh, duren, germany and bluntcloned in the ptz57rt vector with the instaclone pcr cloning kit fermentas inc, vilnius, lithuania. I have 8 expression vectors different tag combinations and im cloning 12 homologs. Infusion advantage pcr cloning kit molecular cloning. 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. Molecular characterization of a copper metallothionein. Thermo scientific clonejet pcr cloning kit efficiently clone pcr products generated with any thermostable dna polymerase using thermo scientific clonejet pcr cloning kit, a positive selection system. Cloning efficiency of the control pcr product into the ptz57rt was.
Any other blunt or stickyend dna fragment can be cloned. Description ta cloning kit designed for cloning directly from a pcr reaction. Original article open access comparative evaluation of. As with the ampicillinresistant pcr script amp cloning kit, the pcr script cam cloning kit is a pcr cloning method that can be performed in 1 hour without adding bases to the primers.
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. 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. Molecular characterization of a copper metallothionein gene from a ciliate tetrahymena farahensis muhammad tariq zahid,1,2 farah rauf shakoori,3 soumble zulifqar,1 ishtiaq ahmed,4 khalid al. For qiagen pcr cloning kit qiagen pcr cloningplus kit pdf 771kb english format. The problem with infusion for my 96reaction expt is the cost of the reaction mix. Overall, we find the strataclone pcr cloning kit highly efficient for cloning of multiple pcr products derived from a single reverse transcription reaction. Strataclone solopack competent cells and puc18 control plasmid.
The tvector pcr product cloning kit is suitable for cloning of pcr products with additional a at 3 end. The clonejet pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific. Choochoo cloning tm kits are highly efficient directional pcr cloning kits for rapid, ligase and restriction enzymeindependent cloning of pcr products. The high quality ta cloning vector ptz57rt is ready to use for efficient ligation with pcr products providing high cloning yields and low background. Gel purify pcr fragment used montage kit, but others probably will work fine tail the purified pcr fragment. Overhangs of this type are generated by taq dna polymerase and other thermostable dna polymerases which lack proofreading activity. I picked 12 white colonies, isolated them and cut with a single cutter enzyme bamhi i cut with bamhi to linearize. Fiveminute cloning of taq polymeraseamplified pcr products. Compare instaclone pcr cloning kit 30 rxns k1214 from thermo fisher scientific on. For my cloning experiment, i first ligated my insert 1. We have reported a set of conserved primers for the identification and.
Infusion advantage protocol overview the table below is a general outline of the protocol used in the infusion advantage pcr cloning kits. Im just about to try plasmid pcr, as per the bieniossek paper. Pseudococcidae muhammad ashfaq, 1 shamim akhtar, 2 saleem akhtar 2, and. Thermo scientific instaclone pcr cloning kit, sufficient for 10. The high quality readytouse ta cloning vector ptz57rt was prepared by linearizing the ptz57r plasmid with eco32i and tailing with. May 03, 2019 the pcr fragment was cloned subsequently using the clonejet pcr cloning kit fermentas. Generacer kit with superscript iii rt and topo ta cloning. Ta cloning technology greatly simplifies traditional restriction and ligation cloning with a onestep cloning strategy that eliminates the need for any enzymatic modifications of the pcr product and not require the use of primers that contain restriction enzyme sites. After plating, i randomly selected 5 colonies from. Instaclone pcr cloning kit free download as pdf file.
Perform pcr reaction as normal using pfu polymeraseif did pcr using taq skip to step 4. Purified nucleotide sequences of the ndna and mtdna genes were subjected to direct sequencing in both directions using the abi prism 3 genetic analyzer and the big dye terminator v. Overhangs of this type are generated by taq dna polymerase and other thermostable dna. Then search the includes part, there you can found ptz57r sequence in. The thermo scientific instaclone pcr cloning kit is a ta system for direct onestep cloning of pcr products with 3da overhangs 1.
Thermo scientific clonejet pcr cloning kit, also available with dh10b cells k123120, is an advanced positive selection system for highefficiency cloning of. After linearization with kpni restriction enzyme, dna concentration was measured and the number of dna copies per milliliter was calculated. Purchase of this product conveys to the purchaser only the nontransferable right under these patents to use the product for research use only by the purchaser. Instaclone pcr cloning kit thermo scientific, poland. Four microgram of lyophilized dna was suspended in 40. The stratagene pcmvscript pcr cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. We used the method of pcr, ta cloning and sequencing. The pcr brochure provides product information on the wide range of dna. The purified pcr product was ligated into vectors according to the instructions of instaclonetm pcr cloning kit fermentas, germany. Design of primers for pcr reaction for quadplex grna cloning the scalability of the multiplex grna cloning kit allows for. Extended protocol and realtime pcr dataset for shrimp species. 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 polymerase. The pdrive cloning vector has a number of useful features designed to facilitate analysis of cloned pcr products. Box ii of the qiagen pcr cloningplus kit containing qiagen ez competent cells should be stored at 70c or.
Pcr products that will be cloned using the qiagen pcr cloning kit should be generated using a thermostable dna polymerase without proofreading activity, such as taq dna polymerase. They have the advantage that their plasmids are smaller, but thats just a small technical issue. Ta cloning relies on the complementary bases of adenine a and thymine t on different dna fragments to hybridize. Molecular characterization of a ctype lysozyme from the. Expression, purification and biological activity assessment. Read below to learn about how to achieve seamless cloning results via topoisomerase cloning, slic, and gibson. These pcr products were used to construct one clone library according to manufacturer of the instaclone pcr cloning kit. What kind of pcr products can be cloned with the qiagen pcr.
Pcr enzyme recommendations for specific applications are listed in table 1. Can the cloning kit be used with inserts containing 5. They allow to clone any pcr fragment into any linearized vector at any location. The pcr fragment was cloned subsequently using the clonejet pcr cloning kit fermentas. Design of a dualpromoter expression vector harboring and. The instaclone pcr cloning kit is compatible with all pcr buffers supplied by thermo scientific.
Briefly, n genes of hcovoc43, hcov229e, hcovhku1 and hcovnl63 were amplified and cloned into ptz57rt thermo scientific, poland plasmids using instaclone pcr cloning kit. The sections below offer stepbystep directions and figure 2a. Pcr based detection of a hyperparasitoid in cotton mealybug, phenacoccus solenopsis tinsley hemiptera. Intragenomic variation in the second internal transcribed. The amplified pcr product was purified using dna purification kit geneall, seoul, korea. Nov 08, 2018 there are many cloning methods that do not require restriction enzymes or ligases. The high quality readytouse ta cloning vector ptz57rt was prepared by linearizing the ptz57r plasmid with eco32i and tailing with single ddt. Jul 11, 2016 four microgram of lyophilized dna was suspended in 40. Qualitative and quantitative assessment of theileria. Instaclone pcr cloning kit thermo scientific, maxima h minus first strand synthesis kit thermo scientific qiaquick gel extraction kit qiagen qiaquick pcr purification kit qiagen subcloning.
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