CIP damages overhangs?
William_D_WARREN at umail.umd.edu
Fri Jan 28 12:32:45 EST 1994
Pat Durham writes:
>Recently I've heard rumors that high concentrations of CIP during
>dephosphorylation prior to ligation can damage overhangs. Has anyone heard of
>this or have any personal experience with this? Reply by email or post in
>this group. Thanks in advance.
>pdurham_pt4 at opal.tufts.edu
I'm not sure about the current commercial products, but early phosphatase
preparations contained low levels of exonuclease contamination. Thus if you
used high concentrations then the ends of your DNA were likely to be nibbled.
THe was around is is to up the pH of the digestion to 9.0 (CIP is also called
Calf Alkaline Phosphatase - for good reason), and to add SDS to 1 or 2%. THe
phosphatase works fine under these conditions but any contaminating
nucleases are inactivated. I still do my phosphatase reactions this way, but
it may be unnecessary depending on your phosphatase supplier.
Hope this helps
William D. WARREN, PhD
Center for Agricultural Biotechnology Email: ww40 at umail.umd.edu
University of Maryland at College Park Phone: (301) 405-7681
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