ABSTRACT
In unit 3, a total of 27 experiments are included. Directional Cloning into Plasmid Vectors describes a standard procedure for cloning DNA fragments with protruding ends. Variables that influence the efficiency of expression of foreign proteins from these and other expression plasmids are discussed in the panel on troubleshooting and optimization of protein expression from an inducible promoter in the Gene Expression in E. coli and Analysis of Gene Products protocol. Assay for β-Galactosidase in Extracts of Mammalian Cells describes the detection of β-galactosidase expressed from reporter vectors transfected into mammalian cells. The assay is both simple and rapid and can be carried out using a visible-light spectrophotometer. The method for Determination of Nucleotide Sequence of DNA by Dideoxy Chain Termination involves the primed synthesis of a complementary radioactive copy of a single-stranded DNA polymerase (Klenow enzyme) in such a way that nested sets of radioactively labelled fragments with 1 common end, but differing at their other end by a specific base, are produced. The base-specific ends are produced by incorporating 2′3′-dideoxynucleoside triphosphates (2′3′ddNTPs) into the growing DNA chain, the ddNTPs lack a hydroxyl group at the 3′ end of deoxyribose, and thus when they are incorporated, further elongation of the chain is stopped. The fragments thus synthesized are separated by high-resolution polyacrylamide gel electrophoresis, which allows fragments differing by a single nucleotide to be resolved, and the sequence can be directly read from the autoradiogram of the gel as dark bands. Preparation of Colony Filters for Hybridization Analysis. The YAC maps are well integrated with the genetic maps of Arabidopsis. Chosen YACs can be used to identify clones in the BAC library by preparing gel-purified YAC chromosomal DNA and hybridizing the isolated DNA to BAC colony filters. The screening of BAC filters by hybridization is described in Hybridization Analysis of Colony Filters. A detailed method for the construction of a library using Arabidopsis genomic DNA is described in Construction of a Binary Cosmid Library from Arabidopsis Genomic DNA. This method can also be used for subcloning BAC DNA and can be adapted for subcloning a YAC, as the source DNA in this case is limiting. In Biolistic Transformation, we have described the genetic transformation of rice by particle bombardment using the PDS-1000/He system.In Protoplast Transformation Using PEG, direct gene transfer to the protoplast exploits the efficient uptake of DNA into the protoplast. It can be enhanced by chemical treatment (for example, with polyethylene glycol [PEG] or by electrical pulses [electroporation]). This method does not require any kind of biological vector. The Agrobacterium -Mediated Transformation of Arabidopsis Root Explants experiment is useful to generate a large number of transgenic Arabidopsis plants and to search for recessive gene mutations using the T-DNA-aided gene-fusion technology. When you read Establishment and Transformation of Arabidopsis cell Suspensions, you will learn an advanced version of a tissue-culture-transformation method, which is based on the co-cultivation of cell suspensions with Agrobacterium that yields an extremely high number of transformants (in the range of 106–107). In Planta Transformation of Arabidopsis by Infiltration, discusses the seed transformation method that was the first successful approach using a large-scale in situ infection of Arabidopsis with Agrobacterium. With this method, a large population of transformants can be raised with less labor than using tissue culture techniques. The PCR Amplification of T-DNA-Tagged Plant DNA Fragments for Automatic DNA Sequencing experiment describes the application of a log range iPCR technique that allows routine amplification and sequencing of T-DNA-linked plant DNA fragments ranging in size from 300 bp to 12 kb. Hybridization Techniques are established and developed on the basis of the denaturation and renaturation of nucleic acids. We have described 3 types of hybridization in this section: Nucleic Acid Hybridization, Northern Hybridization (for RNA only), and Southern Hybridization (for DNA only). This unit also deals with the Assay of the Reported Cat Gene, which emphasizes a reverse-genetics approach. Assay for β-Galactosidase in Extracts of Mammalian Cells is also introduced in form of a protocol to enrich genetic analysis as a part of reverse genetics used in molecular biology.
