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).

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From PMID: 8349594

PCR Protocol    The mixed primers 5'-CCRTGYTGCATNGGYTC-3'
and 5'-ATGAARACNCARGTNGC-3' (N = AGTC, Y = TC, and R = AG
were used for the polymerase chain reaction. Thirty amplification cycles
took place in a 100-ul solution consisting of I uM primers, 0.5 mM
dNTPs, 0.5 pg of P. fluorescens genomic DNA, 5 units of Taq DNA
polymerase, and reaction buffer. The sample was overlaid with 100 pi of
mineral oil and denatured
at 94 "C. Annealing was done over 3 min at 45 %, and polymerization
was accomplished during 3 min at 72 "C. Each polymerization
cycle was increased by 5 s, and the last cycle was extended by 10 min.

Subcloning of PCR Fragment    The pBluescript SK(=) vector was
digested with SmaI and then dephosphorylated with calf intestinal
alkaline phosphatase to block self-ligation. The PCR fragment was
phosphorylated with T4 Polynucleotide kinase and then ligated into the
vector with T4 DNA Ligase.  The reaction
was allowed to proceed for 8 h at -16 "C. The recombinant vector was
transformed into XL1-Blue cells that had been made competent by
calcium chloride treatment (Sambrook et al., 1989). The transformation
was performed according to a Stratagene protocol. Recombinant plasmids
were selected for on LB plates (containing 100 pg/ml ampicillin
and 10 pg/ml tetracycline) that had been spread with 100ul of 100 mM
IPTG and 40 uL of 2% X-Gal (in dimethylformamide) 30 min prior to
plating.

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