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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