Southern conflicts with PCR in knock-in mouse screening

[robobby]

Thanks for clicking this topic!

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?

[admin]

Hey Robobby!,
welcome to the forums.

Hmm... your question is a tough call.

PCR can have contamination, and Southern has its own problems.

Are you using the exact same probe / primers for both techniques?

Could be that you are getting different results as your primer may be amplifying something whereas your probe may be binding to something else...

Try using the same sequence for both and see what happens.

Cheers!