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10.1186/s13036-015-0010-3 http://scihub22266oqcxt.onion/10.1186/s13036-015-0010-3 C4574722!4574722!26388935     free     free     free Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 215.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 249.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 249.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       J+Biol+Eng 2015 ; 9 (ä): ä Nephropedia Template TP {{tp|p=26388935|t=2015. QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids |pdf=|usr=}}{{26388935}} gab.com Text QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids JO: J Biol Eng http://www.kidney.de/pget.php?&tw=1&pmid=26388935 #FOAvip Background: Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result: Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3?-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion: QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. Electronic supplementary material: The online version of this article (doi:10.1186/s13036-015-0010-3) contains supplementary material, which is available to authorized users. Twit Text FOAVip QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids ????J Biol Eng http://www.kidney.de/pget.php?&tw=1&pmid=26388935 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=26388935 #FOAvip English Wikipedia <ref>{{Cite pmid|26388935}}</ref> QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids #MMPMID26388935 Jajesniak P; Wong TS J Biol Eng 2015[]; 9 (ä): ä PMID26388935show ga Background: Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result: Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3?-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion: QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. Electronic supplementary material: The online version of this article (doi:10.1186/s13036-015-0010-3) contains supplementary material, which is available to authorized users. äQuickStep-Cloning: a sequence-independent, ligation-free method for ra(epmc).pdf DeepDyve Pubget Overpricing