uncut vector DNA

Richard J. Dudley rdudley at nrc.uab.edu
Fri Apr 26 16:15:46 EST 1996

For an assymetric digest like that, you shouldn't need phosphatase 
treatment.  Some of your vector is possibly singly digested with either 
enzyme, but that shouldn't be enough to require treatment.  Have you 
tried to gel purify your vector and insert?  Remember to make sure 
you're purifying linear, rather than supercoiled vector.  After that, 
despite all the j/i ratios, load up with insert (v > i leads to a 
problem similar to yours also).  I usually use about 3x i:v.  Finally, 
check your digestion conditions really well, maybe even try another 
buffer.  Good luck!

--- --- ---
Richard J. Dudley
Neurobiology Research Center
University of Alabama School of Medicine
rdudley at nrc.uab.edu

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