Biomedical Engineering Reference
In-Depth Information
Fig.
2
SLiCE cloning of multiple DNA fragments. (
a
) Schematic illustrating multiple-way SLiCE cloning. A three-
way cloning approach is shown. (
b
) Schematic illustrating SLiCE batch cloning (Reproduced from [
4
], with
permission from Oxford University Press)
Our studies indicate that an RecA-independent pathway catalyzes
SLiCE. To improve SLiCE cloning effi ciencies and capabilities, we
established a DH10B-derived strain, termed PPY that was engi-
neered to contain an optimized
prophage Red recombination
system [
4
-
7
]. This strain provides the highest cloning effi ciencies
thus far and can be used for all cloning approaches that are rou-
tinely used in the laboratory [
4
].
SLiCE is a simple and effi cient procedure with the entire pro-
cess consisting of three steps: (1) The preparation of linear vector
and insert fragments containing short end homologies introduced
by PCR with primers having appropriate 5
λ
extension sequences;
(2) the SLiCE in vitro reaction; and (3) the standard transforma-
tion (electroporation or chemical transformation) of recombina-
tion products into suitable host bacteria [
4
].
In this chapter, we describe the preparation of the PPY SLiCE
extract, the maintenance of the PPY strain, and the SLiCE cloning
procedure.
′
