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Train, coach and develop project team members in LIC methods.Project Team Roles and ResponsibilitiesCreating an interdisciplinary team with the right mix of skills is vital to the smooth and successful execution of any project. Team members may be able to cover multiple roles or there may be a sub-team focused on a particular area. Resource allocation depends on the level of expertise team members have, the scope of the project, and budget available.Areas of Expertise

Ligase independent cloning (LIC) is a simple, fast and relatively cheap method to produce expression constructs. It makes use of the 3′–> 5′-activity of T4 DNA polymerase to create very specific 10-15 base single overhangs in the expression vector. PCR products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase as well. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very efficient because only the desired products can form.The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning makes use of restriction enzymes and ligation of DNA in vitro. Restriction endonuclease digestion and ligation increase the complexity of cloning projects, for example by requiring selection of appropriate restriction-sites and inefficient ligation steps. Consequently, several ligation-independent cloning (LIC) methods have since been developed that are simpler, faster, and highly efficient. These strategies rely on the generation of DNA fragments with single-stranded complementary ends to allow directional cloning of any insert, independent of restriction enzymes and in vitro ligation. The most effective and convenient methods include polymerase incomplete primer extension (PIPE) cloning [1], sequence and ligation-independent cloning (SLIC) [2], and overlap extension cloning (OEC) [3], [4] (Figure 1). In this study, we will compare these cloning strategies.Figure 1. Principles of polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC) and overlap extension cloning (OEC).

1. In PIPE, incomplete extension during PCR generates 3′-recessed ends. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. In both these techniques, by amplifying vector and insert with primers containing complementary 5′-tails and mixing the products, the overhangs can anneal and are joined in vivo after transformation into E. coli. In OEC, the insert PCR product acts a megaprimer to generate a nicked plasmid by overlap extension in vitro in a second round of amplification. The nicks are also repaired in vivo. For all techniques, a DpnI digestion step is included to remove plasmid template (not shown). (B) Design of the reporter vector, encoding resistance for ampicillin and kanamycin, and the alpha-fragment of beta-galactosidase. (C) Colonies from ampicillin plates were patched onto kanamycin/X-gal plates to distinguish recombinants from unwanted insert vector or empty pUC18/Kan background colonies.

All three techniques amplify the gene of interest by polymerase chain reaction (PCR). The 3′ ends of the PCR primers are template-specific, whilst the 5′ ends incorporate tails specific for the cloning junction.PIPE relies on the observation that a significant portion of PCR products are incomplete, having 3′-recessed ends, particularly in the absence of a final extension step. SLIC uses a brief treatment of purified PCR products with the 3′→5′ exonuclease activity of T4 DNA polymerase to generate a higher proportion of recessed ends. Cloning is possible due to the complementary 5′ ends of the insert and vector fragments. These are analogous to the much shorter ‘sticky ends’ generated by restriction enzymes. After mixing, these single-stranded overhangs anneal and can be efficiently ligated in vivo after transformation by the bacterial DNA recombination and repair machinery.In contrast, OEC has two rounds of amplification. Firstly, the insert is PCR-amplified using primers with 5′ ends complementary to the target site in a circular destination vector. The 3′ ends of this product (‘megaprimer’) can consequently anneal and amplify the destination vector by overlap extension. This yields a nicked plasmid that is repaired after transformation. A variant of the OEC strategy is utilised by the commonly used QuikChange site-directed mutagenesis kit (Agilent Technologies).In an age of high-throughput molecular biology, there is a need to move away from traditional cloning methods. We believe that LIC methods offer a more robust, efficient means for DNA cloning. Furthermore, variations between strategies suggest that certain techniques may be better suited for particular situations. Consequently, here we characterise and compare the efficiency, convenience, and utility of three major LIC techniques.Develop and implement LIC administrative systems.What Are the Duties of a Personnel Administration Specialist?Most organizations have a division that oversees organizational policies and employee services. This division is often referred to as human resources or office of personnel management (OPM). A personnel administration specialist is a professional who provides support and administrative services to the personnel division. These professionals are also referred to as human resources assistants or specialists. As of June 2010, Indeed.com lists a national average salary of $67,000 per year for this occupation.Department SupportA personnel administrative specialist provides support to the staff of the personnel department by ensuring the department accomplishes assigned responsibilities on a daily basis. This can include coordinating events, arranging meetings and travel plans, creating presentations, preparing reports, answering departmental phones and taking messages for other department staff when they are unavailable.DocumentationAlong with supporting other members of the personnel department, these professionals prepare and maintain all related documentation and employee files for an organization. This includes confidential electronic and paper documentation, such as employment contracts, performance reviews, as well as benefits and compensation information. This documentation is confidential and is required to be secured by the personnel administrative specialist. These professionals often hold all keys and passwords for this information, and access the information for other department staff as requested. This can include creating and compiling personnel reports, and retrieving employee files as requested.Employee RelationsPersonnel administrative specialists are often the gatekeeper of the human resources or personnel department, and assist employees as much as possible unless there’s a need to direct questions and concerns to other department staff. These professionals assist the personnel staff by handling some employees’ relations functions. This can include new employee orientation, enrolling employees in company programs, employee relationship counseling, as well as answering employee questions regarding their benefits and compensation.WHAT IS THE MEANING OF PERSONNEL ADMINISTRATION?Personnel administration is another term for a human resources (HR) job. Duties of a personnel administrator include hiring and training employees, carrying out evaluations and handling employee grievances.DutiesA personnel administrator will usually be charged with the task of keeping employee files up-to-date. They also may be in charge or involved with the hiring process; these duties include interviewing potential candidates and developing training protocol. Workers in personnel administration also perform employee evaluations, counsel employees on their benefits such as health care, and ensure employees are content and comfortable in their place of work by addressing grievances and resolving conflicts between employees.Training and EducationMost workers in personnel administration come to the field with at least a bachelor’s degree. Most universities to not offer undergraduate degrees in HR or personnel administration, but behavioral and social sciences as well as business administration classes are useful. Many employers look for experience, which can be gained through work-study jobs, internships or by performing basic administrative duties for a company.

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