Arking:JCAOligoTutoria23
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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.
- Predict the product
- How big is it? (I get ###### bp)
From PMID:
- Predict the product
- How big is it? (I get ###### bp)