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: 8102367

DNA Manipulations       All DNA manipulations were performed by
standard procedures (17). DNA sequencing was performed using the
dideoxy chain termination method on double strand DNA using
Sequenase version 2.0 (U. S. Biochemical Corp.). E. coli strain DH5a
was used for all DNA manipulations. E. coli strain JM109 was used
for expressing the recombinant protein, and pGEX-3X vector (Pharmacia
LKB Biotechnology Inc.) was used as an expression vector.
pGEX-3X contains an open reading frame encoding glutathione Stransferase
followed by unique restriction endonuclease sites for
BamHI, SmaI, and EcoRI and termination codons in all forward
reading frames. A schematic representation of the strategy for constructing
plasmid pGEX-N is shown in Fig. 1. Using 10 ng of the
sunflower cDNA as a template and oligonucleotide primers RP (5'-
GTAC.. .) and LM3 (5"GATC.. ,) in the polymerase chain reaction
under standard conditions (18), a 155-base pair fragment of DNA
encoding the N-terminal domain of sunflower oleosin was amplified.
Using a restriction site engineered into the LM3 and a restriction site
in the amplified polylinker this fragment was ligated into the BarnHI/
EcoRI sites of expression vector pGEX-3X to produce plasmid
pGEX-N. Plasmid DNA was prepared from several transformants
and checked by restriction digest mapping and DNA sequencing. The
predicted protein product of this in-frame fusion is a glutathione Stransferase-
oleosin/N-terminal domain fusion. The N-terminal region
can be cleaved from glutathione S-transferase via a Factor Xa
JM109.
cleavable sequence (IGYR). This plasmid was then used to transform

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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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