Arking:JCAOligoTutoria23

From PMID: 18045411

Construction of expression plasmid
The coding region of the C. thermophilum glucoamylase
gene was amplified by PCR, using the following oligonucleotide
primers: gla-ep5 5¢-GCGTACGTAGCGGTCGAT
TCCTACATTG-3¢ (SnaBI) and gla-ep3 5¢-GTCGCGGCC
GCTCACCAGTGGTCTTGACCAC-3¢ (NotI). To facilitate
the cloning of the amplified fragment, the sense and
antisense primers contain a SnaBI and a NotI restriction
site respectively. The amplified product was digested with
SnaBI and NotI and ligated to pPIC9K (also digested with
SnaBI and a NotI), producing the P. pastoris secretion
expression plasmid pPIC9K-gla (Fig. 1). The DNA
manipulations were carried out using standard procedures
(Sambrook et al. 1989).