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Quality assurance considerations for use of the FluorImager SI∖R and FragmeNT Analysis software
Author(s) -
Christ Suzanne A.,
Silbiger Richard N.,
Garg Manju,
Franson Susan E.,
Toth Gregory P.
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000301)21:5<874::aid-elps874>3.0.co;2-h
Subject(s) - ethidium bromide , agarose , calibration , fragment (logic) , chromatography , polyacrylamide , calibration curve , electrophoresis , biological system , materials science , analytical chemistry (journal) , chemistry , dna , computer science , microbiology and biotechnology , detection limit , biology , physics , biochemistry , quantum mechanics , programming language
The FluorImager 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.