Hen egg-white lysozyme (HEWL) production by recombinant Aspergillits niger HEWL WT-13-16 from a cDNA under the control of the A. niger glucoamylase promoter was used as a model system. The fungal mycelium was either i...
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Hen egg-white lysozyme (HEWL) production by recombinant Aspergillits niger HEWL WT-13-16 from a cDNA under the control of the A. niger glucoamylase promoter was used as a model system. The fungal mycelium was either immobilized on porous Celite 560 micro-carrier or grown in suspension as pelleted and dispersed forms. The objective was to reduce the protease activity that adversely affects the expressed HEWL. Free suspension culture at uncontrolled pH served as the benchmark. The control of pH during growth at pH 4.0 gave rise to a greater than five-fold reduction of protease activity in suspension culture. An additional 38.5% decrease in protease activity was achieved in mycelial-pellet cultures in comparison to a 40.9% decrease in protease activity obtained with Celite 560 beads in an airlift vessel at controlled pH. The specific HEWL yields were 5.8, 5.0 and 4.1 mg/g dry wt. for the free suspension, mycelial-pellet, and Celite-560-immobilized cultures, respectively.
Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage ...
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Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid copurification of non extended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.
To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high th...
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To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.
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