CDNA文庫（主要內容如下）
· Construction of cDNA Library
· Construction of Genome DNA Library
· Library Screening
Others
· cDNA Library Construction (Michael Blaber's Lab)
Very useful guide and protocol for cDNA library construction
Bidirectional Cloning - Krizman Protocol 1 (Cancer Genome Anatomy Project, NCI)
Protocols for cDNA library construction from micro-bulk and microdissected tissues. Either micro-bulk or microdissected tissues can be the starting material for the cDNA library protocol described here.
Unidirectional Cloning - Krizman Protocol 2 (Cancer Genome Anatomy Project, NCI)
Protocols for cDNA library construction from micro-bulk and microdissected tissues. Either micro-bulk or microdissected tissues can be the starting material for the cDNA library protocol described here.
· cDNA Library Construction (Promega)
Although it's a kit manual, but provides many standard procedures.
o Preparation of Bacterial Culture for Infection
o Ligation of Insert to Vector Arms
o Packaging and Titration of Ligated DNA
o Transfer of Phage to Nitrocellulose Filters for Screening with Nucleic Acid Probes
o Lysate Preparation
Construction of Genome DNA Library (see also Phage, Yeast)
· Construction of Genome DNA Library (Michael Blaber's Lab)
Very useful guide and protocol for genome DNA library construction
· Genomic Cloning Technical Manual (Promega)
Very useful guide for genomic library construction
· Construction of Lambda DNA Library (Promega)
This protocol contains detailed procedures for construction of lambda library, including Preparation of Bacterial Culture for Infection, Ligation of Insert to Vector Arms, Packaging and Titration of Ligated DNA, Transfer of Phage to Nitrocellulose Filters for Screening with Nucleic Acid Probes, Filter Preparation for Screening with Nucleic Acid Probes, Lysate Preparation...
· M13 Library Construction From Cosmids (Whitehead Institute/MIT)
· M13 Library Construction From BAC's or P1's(Whitehead Institute/MIT)
Transfer of DNA from Bacterial Colonies to Nylon Membrane or Nitrocellulose (Donis Keller Lab)
To transfer DNA from bacterial colonies to nylon (or nitrocellulose) membrane so that a library can be screened by hybridization.
· cDNA & Genomic DNA Library Screening (PMCI Research)
· Library Screening using PCR (Crawford Lab)
· Lambda plaque screening (Molecular Genetics Rec Lab)
· cDNA Library Screening (Karl Clark)
o Titer the library to determine the concentration of infectious phage.
o Plate and lift the phage particles.
o Hybridize the immobilized cDNA with labeled probe.
o Pick positive plaques.
o Cycle through steps 2-4 until a single cDNA containing phage is isolated, generally three cycles
· Screening of cDNA and genomic libraries (Atle M. Bones)
o Choice of bacterial strain
o Checking the titer of the library
o Plating out the library
o Taking lifts of the plates, (transferring the lambda phage to nylon membranes).
o Probe labeling
o Filter Hybridization
o Washing of the filters
· Screening of libraries with antibodies (Molecular Biology Laboratory (Dept. of Clinical Pharmacology, University of Berne, Switzerland)
The colony blot procedure is used to screen a library with an antibody for the expression of an antigen by a clone.
Filter hybridisation with radioactively labelled probe: Denhardt method (Molecular Genetics Rec Lab)
Filter hybridization with digoxigenin labelled probes (Molecular Genetics Rec Lab)
· Colony/Plaque Blotting (Gimila's Lab)
· Colony Hybridization (Vesicle Trafficking)
· Phage Titre and Solution Preparation (Gimila's Lab)
· Plaque Lifts (Gimila's Lab)
Amplifying Plasmid Libraries (LTI)
Plasmid libraries may be amplified using several different methods