I need to measure total cellular RNA and protein in ethanol fixed baculovirus infected Sf9 cells. I found a protocol by Crissman (1982), Cytometry 3:(2) 84-90 for cellular DNA and protein using PI/FITC and RNase treatment and think that substituting DNase for RNase and a DNase buffer for PBS may just do the trick. Has anyone used this method or have an opinion of its usefulness or alternatives? In addition... PI/DNase/RNase controls indicate that I'm not getting rid of ALL the DNA using DNase A at 1.5mg/ml during a 40min digestion! The mean FL3(630nm) channel measured on a linear scale after DNase treatment and PI staining is also rather eratic from one sample to another in a time series and repeating the analysis on 15 fixed samples a day apart seemed to give me only a very weak correlation between duplicates of the mean FL3 channel. What could I be doing wrong? If it is the residual DNA effect how do I remove it? Regards Matt _______________________________________________________ Matthew Rosinski The University of Queensland Department of Chemical Engineering College Rd St Lucia Q 4072 Australia Ph: (07) 3365 8392/4352 _____________________________________________________
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