Development of a microfluidic chip-based plasmid miniprep.
Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10(5) times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.
Northrup
Victoria A
VA
Department of Medical Genetics, University of Alberta, Edmonton, Canada.
Backhouse
Christopher J
CJ
Glerum
D Moira
DM
D002876:Chromosomes, Bacterial / Q000737:chemistry*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004867:Equipment Design; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics; D044085:Microfluidics / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D014170:Transformation, Genetic
D004269:DNA, Bacterial