UV Shadowing

UVshadowingisatechniqueforvisualizingnucleicacidsseparatedonacrylamide/ureagels.ThetechniqueutilizesshortwaveUVlight(254nm)andafluor-coatedTLCplate.WerecommendUVshadowingasthemethodofchoiceforgelpurificationofnonisotopicprobessynthesizedwithAmbion"sMAXIscript®Kit.ThealternativetoUVshadowingisstainingwithethidiumbromideoracridineorangeandrequiressubsequentextractionofthedye.ThedetectionlimitofUVshadowingisapproximately0.3µgofnucleicacid.

Procedure

1. Afterelectrophoresis,removeoneoftheglassplatesandcoverthegelwithplasticwrap.
2. Placethegel,plasticwrap-side-down,onaflatsurfaceandslowlyremovetheotherglassplate.
3. (optional)Coverthegelwithasecondsheetofplasticwrap.
4. Inadarkenedroom,placetheplastic-wrappedgelontopofthefluor-coatedTLCplate.
5. Visualizenucleicacidbandsbyshiningahand-heldUVlightsource(254nm;shortwave)onthesurfaceofthegel.
6. NucleicacidwillappearasdarkpurplebandswhiletheTLCplatewillappeargreen.Note:ThexylenecyanolandbromophenolbluedyespresentinmostgelloADIngbuffersmayalsoappearasdarkbandsduringUVshadowingandaresometimesmistakenfornucleicacid.Werecommendthatyourunanextralaneofloadingbufferaloneinordertoeasilydistinguishbetweenthesedyesandashadowcausedbynucleicacids.
7. Markthebandtoberemoved.
8. Cutoutandremovethebandcorrespondingtothedesirednucleicacid.

Figure1.MicrogramDilutions(asLabeled)ofanRNATranscriptSeparatedonaDenaturing0.75mmThick5%Acrylamide/8MUreaGel.Thewelldimensionswere6mmwide.Afterelectrophoresis,thegelwasremovedfromtheglassplatesandUVshadowedfromabovewithhand-held,shortwaveUVsource.TheimagewascapturedonanAlphaInnotech500imagingsystem.