#Serial cloner preimer efficiency manual

As a result, the mainstream strategy for the construction of recombinant expression vector includes two cloning steps: PCR cloning (cloning of PCR amplified target gene into cloning vector) and restriction enzyme mediated subcloning of target gene from cloning vector into expression vector. PCR has been indispensible for gene cloning because it can provide target gene fragments in vitro in large amount and short time without the limitation of restriction sites. Cohen initiated the genetic engineering era. pOmni developed in our study provides a powerful tool for gene cloning and expression, and is of special value for researches in which both prokaryotic and eukaryotic expression of a target gene are necessary.Д®xpression of a target gene in prokaryotic cell or/and eukaryotic cell has become a common research goal or a basic experimental procedure for profound researches of genes of interest since Herbert Boyer and Stanley N. The cloning capability and reliability of selection-free PCR recombination cloning with pOmni were validated through cloning of 6 DNA fragments with length from 315 to 4557В bp, and the dual-expression function of the vector was verified through the cloning and expression of EGFP in E. Furthermore, the target gene cloned in pOmni is ready to be high-efficiently expressed in either Escherichia coli cells or eukaryotic cells because of the elaborate design of compatible T7 promoter and CMV promoter expression elements in the vector. In this study, we report the design, and construction of an omnipotent expression vector pOmni, with which a target gene can be easily cloned through innovative selection-free PCR recombination cloning strategy with only one pair of primer and two times of PCR in one work day, without using any restriction enzymes, ligase and other DNA modifying enzymes. A variety of ligase-independent PCR cloning strategies have been developed, but they either involve complicated PCR procedures or need other DNA modifying enzymes.

#Serial cloner preimer efficiency manual

In Vitro Mutagenesis: Methods and ProtocolsВ guides readers through methods for gene and genome editing, practical bioinformatics approaches for identifying mutagenesis targets, and novel site-directed and random mutagenesis approaches aimed at gaining a better understanding of protein-protein and protein-cofactor interactions.В Written in the highly successful Methods in Molecular BiologyВ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.ДЄuthoritative and cutting-edge, In Vitro Mutagenesis: Methods and ProtocolsВ aims to provide a highly accessible and practical manual for current and future molecular biology researchers, from the beginner practitioner to the advanced investigator in fields such as molecular genetics, biochemistry, and biochemical and metabolic engineering.The conventional procedure for the construction of recombinant expression vector of a target gene includes PCR cloning and restriction enzyme mediated subcloning, which is time-consuming and sometimes troublesome because of the inefficiency of ligation. This volume provides a wide variety of updated and novel approaches for performing in vitroВ mutagenesis using such methods as genome editing, transposon (Tn) mutagenesis, site-directed, and random mutagenesis. In vitro mutagenesis remains a critical experimental approach for investigating gene and protein function at the cellular level.