69281  DNase Shotgun^® Cleavage Kit

Random, double-stranded cleavage of any DNA for shotgun cloning

The DNase Shotgun® Cleavage Kit is designed to generate random DNA fragments from any DNA sample in preparation for cloning. The sample DNA is cleared with DNase I in the presence of Mn²⁺, which causes random double-stranded cleavage of the DNA molecule (Anderson 1981). The DNase I in the kit is specially prepared in a buffer lacking Mg²⁺ to prevent single-strand nicking.

Fragments of almost any size can be generated by adjusting the amount of enzyme and/or time of reaction. Specific size ranges can be isolated by agarose gel electrophoresis, and the ends repaired using a DNA polymerase (e.g., to generate blunt ends treat with T4 DNA polymerase in the presence of dNTPs). The Single dA Trailing Kit repairs DNA ends and adds a 3′-dA residue. The processed DNA fragments can be easily cloned into any vector with single 3′-dT or 3′-dU overhangs, such as one of the Novagen AccepTor™ Vectors. Advantages of this cloning strategy include: prevention of tandem inserts, low nonrecombinant background, and no requirements for special linkers or additional fractionation steps. This patented approach is used to generate protein domain expression libraries with the Novagen NovaTope® System.