A RAPID AND SIMPLE METHOD FOR SCREENING LARGE NUMBERS OF RECOMBINANT DNA CLONES

摘要

Screening recombinant DNA clones from cDNA library or transformation experiments is a regular task in molecular biology research; the routine alkaline extraction procedure is tedious and time consuming (90-120 min for 12 samples), and unsuitable for screening large numbers of clones from these experiments, especially when no blue-white indicator (l-complementation of lacZ gene) exists in recombinant clones. Some commercial plasmid preparation kits are more rapid (20-60 min for 12 samples) and simple in plasmid extraction; however, they are costly on a daily basis. Therefore, a simple, rapid and economic method for screening recombinant DNA clones is needed. Here we present an extremely simple, rapid and economic procedure of plasmid DNA preparation for screening recombinant clones from cDNA library or transformation experiments based on a modified alkaline lysis method. In this method, bacterial colonies or cultures were directly lysed in 2% NaOH solution, and then recombinant plasmids in the supernatant were visualized quickly by agarose gel electrophoresis. The entire process, from Escherichia coli colonies or cultures to plasmid DNA, takes only about 12-15 min for 12 samples, which is the quickest method for plasmid preparation in our best knowledge. Hundreds of recombinant clones with no easy screening method can be rapidly identified in several hours using this method. Our approach is extremely rapid and simple to screen large numbers of recombinant clones from bacterial colonies or bacterial cultures of cDNA library or transformation experiments. It is especially suitable for identifying those high copy recombinant vectors without blue-white color reaction such as pDNR-LIB, pPICZlA, etc. In addition, this protocol is also suitable for extracting plasmids and screening recombinant DNA clones from bacterial cultures. Our data have indicated that plasmids extracted by our method could meet the criteria for restriction enzyme digestion after neutralization.