Efficiency of DIG Labeling kit (Roche) - (May/07/2007 )
I am trying to determine the copy no. of an transgene using cotton genomic DNA (30ug). Roche DIG DNA labeling and detection starter kit-II was used for the said purpose. Some how no results were obtained except +ve control where plasmid DNA was used as template. some one told me that only radioactive P32 will work. Is it true or is it posible to get result of interest thru Roche DIG DNA labeling and detection starter kit-II. please guide me.
This depends crucially on the method used for detection. With chemiluminescent detection and sensitive readout (film or chillled CCD imager) detection can be about as good as with P32. I don't, however, see how you can do copy number detection this way, with only 2x difference (max) between samples. This will be very difficult to quantitate with any sort of blot or dot-blot technique. Quantitative PCR is the way to go. FYI I have found that DIG labeling by spiking normal PCR reactions with dig-dUTP works very well, and avoids a lot of uncertainty in other label protocols. You have to use an enzyme that will incorporate dUTP, such as Taq.