Cloning procedures

Isolation of plasmid DNA, transformation, transfection and cloning procedures were performed using standard techniques (Sambrook et ut/., 1989). 

Marker excision by site-specific recombination Very clean excision, small footprint Complex cloning procedure. Requires additional transgene encoding Cre recombinase 39... 

Wendland, J. 2003. PCR-based methods facilitate targeted gene manipulations and cloning procedures. Current Genetics 44,115-123. 

E. coli Rapid cloning procedure High expression levels Easy scale-up Toxicity of foreign membrane proteins Lack of post-translational modifications... 

Definition of the background activity of microinjected oocytes is a clear prerequisite for any functional cloning procedure, as a positive signal needs to be distinguished from the endogenous transport activities. Key characteristics of the carrier, such as a dependency on sodium ions or a specific inhibitor are very helpful. In addition, unspecific binding of the radioactive transporter substrate to the oocyte membrane needs to be examined carefully. 

Cloning procedure (in bacteria) Recombinant plasmids transfected into bacteria Antibiotic resistance used to select bacteria with recombinant plasmids Blot growth plate and probe with P-DNA for gene, or with Tantibody for protein... 

The same cloning procedure has also been used for obtaining cell variants of decreased sensitivity [54], In this case S49.1 subclones were isolated which grew in the presence of much lower concentrations, i.e., 10 nM dexamethasone. These variants turned out to contain significantly reduced levels of glucocorticoid receptors with unaltered affinity for the steroid. This observation suggests that receptors are not only a necessary requirement for hormone action but also play a quantitative role. A relationship between cellular receptor content and the extent of hormonal sensitivity has in fact been seen in a series of mouse lymphomas of independent origin [52]. 

Techniques for the production of individual cDNAs, cloned in bacteria at the rate of one cDNA per bacterium, were multiplexed to produce cDNA libraries , large collections of cDNAs, simply by repeating the cloning procedure with complex cDNA mixtures. These complex cDNA mixtures, now in bacteria, can be surveyed by retrieving and sequencing each individual cDNA, giving a faithful representation of the abundance of each cDNA in the original mixture if sufficient cDNAs are sequenced. 

Fig. 4.23. Route for cloning double-stranded cDNA from reverse transcripts of viral RNA and subsequent sequencing (not using M13 cloning procedure).

For monoclonal antibodies, the submission should include a detailed description of the development of the monoclonal antibody, including characterization of the parent cells, donor history for human cells, immunogen, immortalization procedures, and cell cloning procedures. 

Tanaka H, Ohshima N, Hidaka H. Isolation of cDNAs encoding cellular drug-binding proteins using a novel expression cloning procedure Drug-western. Mol. Pharmacol. 1999 55 356-363. 

FlCURE B-5 Detail of the cloning procedure. The steps involving reverse transcriptase, UNA polymerase i, and terminal transferase are shown. The overall goal is to pr uce dsDNA suitable for insertion into the cloning plasmid, using inRNAa the starting material. 

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