o. van-ham wrote:
>> I'm developing a protocol that includes a step where DNA needs to be cut
> randomly, leaving blunt ends. I initially digested the DNA with DNase I
> and cut out the band corresponding to linear DNA. When I sequenced my
> final clones, it turned out that many of the termini had been chewed
>>> Does anyone have any suggestions? If so, please let me know.
How about partial digestion with four-base cutters like HaeIII, ThaI or
AluI? Should give you a pseudo-random set of fragments. If you're using
supercoiled plasmid DNA as the starting point, ethidium bromide works
well as a means of controlling the amount of digestion. [Parker, R.C.
(1980). Conversion of circular DNA to linear strands for mapping. Methods
in Enzymology 65, 415-426.].
Otherwise, sonication followed by end repair is what people use (or used
to use?) for sequenceing projects.