Plasmid library

Add 240 pi of 50% PEG-3350, 36 pi 1.0 M LiAc, 50 pi of herring testes carrier DNA (2 mg/ml) x pi library plasmid DNA (0.1 to 10 pi), and sterile water to a final volume of 360 pi. It is important that the components are added in the exact sequence listed above. Vortex vigorously until the cell pellet has been completely mixed. This usually takes about 1 min. Incubate at 30°C for 30 min. 

Colonies that grow on the CM(-leu-his-trp) plates and that express high levels of P-galactosidase are picked and grown overnight in CM(-leu) medium. This maintains selection for the library plasmid and removes the selection for the bait plasmid. Therefore, a small percentage of yeast will lose the bait plasmid, but retain the prey plasmid. These are readily identified by the following protocol. 

An alternative method to examine specificity is in a mating assay. Here, a strain of yeast of the opposite mating type, which harbors the individual unrelated bait constructs is crossed with the strain carrying the isolated library plasmid This is a more efficient method to rapidly screen large numbers of potential clones. 

Fig. 4. Steps in making a cDNA library. Cellular mRNA is used as a template to make a complementary DNA. This cDNA is then ligated to a plasmid,...

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

Following several cycles of mutagenesis using the E. coli XLl-Red mutator strain and transformation of the plasmid library into E. coli, a total of about 150 000 bacterial colonies were assayed for activity using a colorimetric prescreen [100]. The best mutant Asn336Ser showed a 47-fold increase in activity and a 5.8-fold enhancement... 

Figure 2.3 Metagenomic cloning experiments. Isolation of genomic DNA directly from environments (soil, plants, mixed environments or thermal-vent worms are the examples Illustrated here) can recover DNA fragments which could encode for enzymes. The DNA fragments can be ligated to plasmids or DNA linkers, and then subjected to functional screening (expression cloning) and/or sequence analysis. Amplification by PCR can sometimes be used to yield libraries enriched with clones containing selected sequence motifs relating to families of enzymes...

Bacterial colonies containing plasmids (library ot transform)... 

PCR reaction using natural dNTPs. However, there are some drawbacks to epPCR. Generally, the technique produces libraries of DNA fragments which need to be ligated into expression plasmids (this can be a limiting step), and some of the methods do not produce random mutations. 

---