Titlebook: Genetic Library Construction and Screening; Advanced Techniques R. Curtis Bird,Bruce F. Smith Book 2002 Springer-Verlag Berlin Heidelberg

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Strategies for cDNA Cloning and Mapping RNA Transcripts as determining any splicing (including alternative splicing) patterns can lead to erroneous conclusions. Investigations such as the identification of promoters or enhancers also require that the primary structure of the transcript be previously determined.

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Site Directed Deletion, Insertion, and Substitution using PCR significant improvements have been made in using thermolabile polymerases as enzymes and double-stranded DNA as templates, by integrating strong selection methods, e.g., Unique Site Elimination based on restriction digestion at a site unique to the wild type (.), into the mutagenesis methods.

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Yeast One and Two Hybrid cDNA Cloningught to the promoter containing the GAL4 DNA binding sites and will activate the transcription of reporter gene HIS3 or lacZ. The two-hybrid system can be used to clone cDNA encoding a novel protein that interacts with a known protein (bait) in yeast. It can also be used to study protein-protein interactions between two known proteins.

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Long RT-PCR Cloning — Amplification of Full-Length Enterovirus Genomesnd Taq polymerases such as rTth DNA polymerase, XL (PE Applied Biosystems), Deep Vent DNA polymerase (New England Biolabs) and the polymerase mix provided with the Advantage 2 PCR Enzyme System (BD Biosciences, Clonetech, UK) which have high fidelity rates and can generate much longer PCR products.

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Construction of cDNA Libraries from Small Quantities of Total RNA Using Template Switching Catalyzedategies have been developed to add a determined sequence (anchor) at the 3′ end of the first-strand cDNA. These strategies include: (1) oligo (dG) or oligo (dA) tailing by terminal deoxynu- cleotidyltransferase (.; .); ( 2) the use of T4 RNA ligase to covalently attach a single-stranded (ss) anchor

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