Southern conflicts with PCR in knock-in mouse screening
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I am working on a single mutation Knock-in mouse project. Usually southern hybridization is the golden rule to screen the ES cells and first generations of mice. We found some positive Knock-in ES cells. From one of these cells, we even got mice. However, when we amplify the segments flanking the mutation and sequence the PCR product, many of these ES cells and all mice turned out to be wild-type.
Does any one know why this happens? Should I trust southern or PCR in screening?
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