Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.USER-Derived Cloning Methods and Their Primer Design
Uracil excision-based cloning through USER™ (Uracil-Specific Excision Reagent) is an efficient ligase-free cloning technique that comprises USER cloning, USER fusion, and USER cassette-free (UCF) USER fusion. These USER-derived cloning techniques enable seamless assembly of multiple DNA fragments in one construct. Though governed by a few simple rules primer design for USER-based fusion of PCR fragments can prove time-consuming for inexperienced users. The Primer Help for USER (PHUSER) software is an easy-to-use primer design tool for USER-based methods. In this chapter, we present a PHUSER software protocol for desi...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Hierarchical Ligation-Independent Assembly of PCR Fragments
The emerging field of synthetic biology requires novel cloning techniques that allow the rapid assembly of multiple expression units to build artificial genetic circuits. Here, we describe a rapid, flexible, and cost-efficient cloning method that requires only standard laboratory equipment and skills. Our technique relies on the 3′–5′ exonuclease activity of T4 DNA polymerase to generate 20 nt single-stranded DNA overhangs that allow annealing and ligation-independent cloning (LIC) of four DNA fragments in one tube. The resulting intermediate-size constructs can be reused to hierarchically assemble constr...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Quick and Clean Cloning
Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that conta...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Plasmid Construction by SLIC or Sequence and Ligation-Independent Cloning
Sequence and ligation-independent cloning (Nat Methods 4:251–256, 2007) is a powerful tool for the construction of multi-fragment complex plasmids in a simple and efficient manner. Plasmids consisting of 6–7 DNA fragments can be assembled in a single day, with additional 2 days for screening and extraction. SLIC requires PCR products with overlapping regions of 30–40 bp at the 5′ and 3′ ends, T4 DNA polymerase, and an optional RecA protein for construction. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
BioBrick Assembly Standards and Techniques and Associated Software Tools
The BioBrick idea was developed to introduce the engineering principles of abstraction and standardization into synthetic biology. BioBricks are DNA sequences that serve a defined biological function and can be readily assembled with any other BioBrick parts to create new BioBricks with novel properties. In order to achieve this, several assembly standards can be used. Which assembly standards a BioBrick is compatible with, depends on the prefix and suffix sequences surrounding the part. In this chapter, five of the most common assembly standards will be described, as well as some of the most used assembly techniques, clon...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis
This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a program to design oligonucleotide sets for long sequence assembly by the ligase chain reacti...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
j5 DNA Assembly Design Automation
Modern standardized methodologies, described in detail in the previous chapters of this book, have enabled the software-automated design of optimized DNA construction protocols. This chapter describes how to design (combinatorial) scar-less DNA assembly protocols using the web-based software j5. j5 assists biomedical and biotechnological researchers construct DNA by automating the design of optimized protocols for flanking homology sequence as well as type IIS endonuclease-mediated DNA assembly methodologies. Unlike any other software tool available today, j5 designs scar-less combinatorial DNA assembly protocols, performs...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Seamless Ligation Cloning Extract (SLiCE) Cloning Method
SLiCE (Seamless Ligation Cloning Extract) is a novel cloning method that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (15–52 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from bacterial artificial chromosomes or other sources. SLiCE is highly cost-effective and demonstrates the versat...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Application of In-Fusion™ Cloning for the Parallel Construction of E. coli Expression Vectors
In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Combinatorial Assembly of Clone Libraries Using Site-Specific Recombination
Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clo...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR
We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase. This cloning technology can be applied to a few common Escherichia coli hosts (e.g., BL21(DE3), DH5α, JM109, TOP10). The protocol includes three steps: (a) linear DNA fragments (i.e., the insert DNA and the vector backbone) with two overlap ends were generated by regular high-fidelity PCR, (b) the DNA multimers were generated based on these equimolar DNA templates by using prolonged overlap extension PCR (POE-...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Minimum GC-Rich Sequences for Overlap Extension PCR and Primer Annealing
PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20–30 nucleotides long and identical to a part of template DNA. However, primers often contain additional sequences at their 5′ ends, which are restriction enzyme sites, recombination targeting sequences, or overlap sequences for fusion PCR. When these additional sequences are attached to their annealing sequences, the annealing sequences can be shortened. Here, we describe universal GC-rich annealing sequences useful for overlap extension PCR and sim...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
FX Cloning: A Simple and Robust High-Throughput Cloning Method for Protein Expression
The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a straightforward and efficient tool. It is dedicated to the cloning of open reading frames (ORFs) with the final aim of expressing the corresponding proteins. FX cloning combines attractive features of established high-throughput cloning methods that were thus far not unified in one single method. It facilitates the subcloning of a sequence-ve...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy
GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named “GB parts” and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4–5 TUs are routine...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
High-Pressure Freezing and Freeze Substitution of Arabidopsis for Electron Microscopy
The objectives of electron microscopy ultrastructural studies are to examine cellular architecture and relate the cell’s structural machinery to dynamic functional roles. This aspiration is difficult to achieve if specimens have not been adequately preserved in a “living state”; hence specimen preparation is of the utmost importance for the success of any electron micrographic study. High-pressure freezing (HPF)/freeze substitution (FS) has long been recognized as the primer technique for the preservation of ultrastructure in biological samples. In most cases a basic HPF/freeze substitution protocol is su...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
