Genescan Tutorial

Fragment Analysis at the CSLOregon State UniversityMarker discovery, e.g. database searches,sequencing, preliminary screeningw/[F]dntpsDesign and purchase labeled primersResearcher*Perform PCR single- or multi-plex ;Post-PCR multiplex if desiredSubmit sample sheet and samplesDownstream analysisDiscuss researcher’s goals and provideguidance for obtaining good resultsPour slab gel for the ABI 377CSLPrepare and aliquot master mixDenature samples, load and run gelPost-run preliminary analysis:GeneScan and GenotyperIn the core facility (CSL) at Oregon State University the researchers bring thesamples which they have developed, amplified, diluted and multiplexed. Weprepare and add the master mix (internal lane standard), run the samples on aslab gel, convert the raw data to numbers and return the data to the researchersfor further analysis.Nature Biotechnology (Feb.2000) Vol 18: pp233-234 details a method toreduce costs for fluorescent labeling of PCR primers. It is a way that one ofthe researchers labels both strands of the samples run on a non-denaturing(SSCP) gel.1Spectral Overlap - Importance of MatrixBefore applying matrix After applying matrixNote:On the leftthe lane withFAM isshowingfalse colors(other thanBLUE)On the rightthe same laneis showingonly BLUEbecause thematrix haseliminatedthe falsecolors. Understanding the effect of spectral overlap, using matricies to overcome orreduce spectral ...