Manual: StrataPrep® Plasmid Miniprep Kit

TROUBLESHOOTINGObservationThe plasmid DNA is contaminatedwith chromosomal DNAThe plasmid DNA is notsupercoiledThe cells are not lysed with theaddition of solution 2The DNA floats out of the wells ofthe agarose gelSuggestionChromosomal DNA is sheared by vortexing the tube to mix the contents afteradding solution 2 or solution 3. Mix the contents of the tube by gentle inversion;do not vortexPlasmid DNA is nicked by nuclease contamination. Include the wash procedure instep 12 in the protocolSolution 2 is degraded by reaction with air over time. Keep the container ofsolution 2 capped tightly before and after use; make a fresh solution if necessaryMake sure that all the wash buffer is removed from the microspin cup beforeadding the elution buffer to avoid ethanol contaminationPREPARATION OF REAGENTSElution Buffer10 mM Tris baseAdjust pH to 8.5 with HClor10 mM Tris base1 mM EDTAAdjust pH to 8.0 with HClorSterile ddH 2 OSolution 20.2 M NaOH1% (w/v) SDSNote Store tightly capped beforeand after use.Solution 150 mM Tris HCl (pH 7.5)10 mM EDTA50 µg/ml of RNase A2× Wash Buffer10 mM Tris HCl (pH 7.5)100 mM NaCl2.5 mM EDTAREFERENCES1. Birnboim, H. C. and Doly, J. (1979) Nucleic Acids Res 7(6):1513-23.2. Vogelstein, B. and Gillespie, D. (1979) Proc Natl Acad Sci U S A 76(2):615-9.ENDNOTESStrataPrep ® is a registered trademark of Stratagene in the United States.6 StrataPrep ® Plasmid Miniprep Kit