Southern Blot Using Copy Standards, Example

Southern blot for detection of transgene integrations and copy number, example.


10 micrograms per lane of tail DNA was digested with BamHI and EcoRI, then electrophoresed on 1.2% agarose, and alkaline capillary transferred to Hybond N+. The blot was hybridized with random prime-32P-dCTP-labeled probe from the transgene. This probe hybridizes with 6 and 5 kb endogenous bands seen in each lane, and the 2 kb transgene band seen in the 1X, 5X, and 10X transgene DNA-spiked tail DNA lanes, two MLC-Sno control lanes containing tail DNA from another, single-copy, transgenic line, and four new transgenic mice identified in this Southern blot. Inclusion of the copy standards allowed us to calculate the approximate number of transgenes integrated for each founder as one for #10054, six for #10116, sixteen for #10050, and sixty for #10052. Thus, four out of 30 mice screened (13%) were transgenic, which is lower than our recent track record of 20% transgenic.