Interactive control of pulsed field gel electrophoresis via real time monitoring.
The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge-coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis is possible throughout the run via this detection scheme. The use of absorption removes the need for staining or radioisotope labelling, thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without destaining. The optimization of buffer concentration, electric field strength, temperature, agarose concentration, as well as pulse duration can considerably reduce total run time. For example, DNA from 2 to 850 kb can be separated in 3 h on a 7-cm gel with interactive control of the pulse time, which is 10 times faster than using a constant field program.
McGregor
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Ames Laboratory-USDOE, Iowa State University 50011.
Yeung
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D004247:DNA / Q000032:analysis; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D004247:DNA