Morten Lukacs wrote:
2).Is if sufficient to purify the phosphatase treated DNA by simply load a
on an agarosegel, and afterwards just purify the DNA from the gel....
or is it necessary to do a phenol extraction and ethanol precipitation ???
I routinely treat vector DNA with CIP in 20 microliter rxns and load
straight to preparative AGE. This approach seems to work well, but I've
never actually compared it to other techniques.
Two possible advantages: (1) One-step, with no loss of DNA during phenol
extraction; (2) vector is gel-purified and, thus, contains no
contaminating uncut portions.
If you go this route, I recommend Jim Graham's protocol for recovery of DNA
from agarose gels archived in 9304, "Consistent subcloning from agarose
Dept. Biochem. and Mol. Biol.
Okla. St. U.
Stillwater, OK 74074 USA
shartson at bmb-fs1.biochem.okstate.edu