Quality of assurance considerations for use of the Fluorlmager SI and fragmeNT analysis software.
The Fluorlmager SI (FSI) from Molecular Dynamics is one of several scanning instruments available for the detection of fluorescent emissions associated with DNA samples in a variety of matrices (agarose and polyacrylamide gels, membranes and microplates). In our laboratory, we measured the electrophoretic mobility of randomly amplified polymorphic DNA (RAPD) fragments stained with ethidium bromide in agarose using the FSI to scan gels and the associated Molecular Dynamics software (ImageQuaNT, and FragmeNT Analysis) for analysis. Initial scans and analyses resulted in inconsistent band detection across the same gel and across several scans of the same gel. To determine the best types of calibration for the instrument, several factors were considered and then evaluated. Tests of calibration acceptability were also evaluated. Band detection by FragmeNT Analysis was improved following optimization of matrices and parameters used in calibration and experimental scans. In addition, use of software templates for analysis and modifications in the staining procedure, which have resulted in decreased instrument associated variance, are discussed.
Christ
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United States Environmental Protection Agency, National Exposure Research Laboratory, Ecological Exposure Research Division, Cincinnati, OH 45268, USA. christ.suzanne@epa.gov
Silbiger
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Garg
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Franson
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Toth
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D002138:Calibration; D004247:DNA / Q000032:analysis; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation*; D011786:Quality Control; D012984:Software; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D004247:DNA; D015247:Deoxyribonuclease HindIII; D004996:Ethidium