Cloning plasmids

Vectors for recombinant DNA and cloning Plasmid pBR322 Restriction site Replication origin Resistance to antibiotic(s) Expression vector also requires Promoter Shine-Dalgarno sequence Other vectors phage, YACs... 

Cloning-Ready/ Entry Clone plasmid minipreps... 

The concentration of an Entry clone plasmid should be kept at a low level (1-10 ng/pi) in the LR reaction. A high amount of an Entry clone proved to be adverse, contrary to our expectations. Similarly, the concentration of the destination vector should be kept low. In contrast to the LR reaction, it would be better to use the insert DNA (PCR products) at a high concentration in the BP reaction. 

Figure 26-7 Genetic map of cloning plasmids pBR322 and pUC18. Abbreviations ori, origin of replication Amp1, ampicillin resistance gene Tet1, tetracycline resistance gene. Other abbreviations are for sites cleaved by specific restriction endonucleases, a few of which are defined in Table 26-2. The nucleotide sequence numbers and directions of transcription are also indicated. Reproduced by permission of Amersham Pharmacia Biotech Inc.

Cloning was confirmed by restriction digestions and agarose gel electrophoresis. It demonstrated that the newly cloned plasmid was successfully transformed into the E. coli XLl-Blue, HMS174, DH5a and demonstrated that the construction of the recombinant E. coli with plasmid pUC19 containing the entire pha operon was successful. 

Figure 6.28. Microinjection of DNA. Cloned plasmid DNA is being microinjected into the male pronucleus of a fertilized mouse egg. 

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. 

A typical affinity maturation project using yeast cell surface display is divided into several steps. First, the variable (VH and VL) domains of the parent hybridoma clone are identified. Second, the VH and VL genes are cloned into a display vector for expression as a scFv fragment on the cell surface. Third, the binding characteristics of the WT scFv are determined. Fourth, diverse variegated populations for selection are created by mutagenesis of the parent clone plasmid DNA. Fifth, clones with improved affinities are enriched from the mutant library after application of selective pressure. Sixth, selected clones are sequenced and... 

Fig. 3.6 Southern blot indicating the copy number of DNA encoding the light chain in different IgC expressing PER.C6 clones. Plasmid copies are measured to a standard comprising a known amount of plasmid DNA in a background of human chromosomal DNA.

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