A PCR-after-ligation method for cloning of multiple DNA inserts.
Here we present a novel and simple PCR-after-ligation method for efficient assembly of multiple DNA inserts. After initial ligation of multiple inserts and vector, the ligation mixture is used as template for a PCR using a pair of primers flanking the cloning sites on the vector. The fragment with correct size is gel purified and inserted into the vector by conventional two-way ligation. With this method, a recombinant plasmid containing four DNA inserts was correctly constructed. As a control, all of the constructs obtained directly from DNA ligation were found to be self-ligation of the vector.
An
Yingfeng
Y
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, People's Republic of China.
Wu
Wenfang
W
Lv
Anguo
A
D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*
D004247:DNA