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Double Digestion Problem!!!
I have (at least I think I have) modified my BAC. The problem that i am having is that when i pcr out a segment of my vector from the BAC I get a band that is exactly where it shoud be (forward primer is 5' of insert and reverse primer is within my insert). When I double digest (Sal1 and Not1) I dont see the appropriate shifts that I expect which tells me that my shuttle vector did not get inserted into my BAC?? My BAC is 190kb and I run the gel using pulse field electro. Any suggestions as to what is going on???
|digestion , double , problem|
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