Did anyone transform un-digested pPICZalpha-A (circular DNA) which was inserted a desired gene into Pichia pastoris using electroporation?
According to User Manual's Invitrogen (EasySelect Pichia Expression Kit), they suggested that we should use 50-100 micrograms circular DNA to electroporate into Pichia. In your experiments, how many micrograms of un-digested plasmids did you use? Thank you very much for your help!
Hi there,
Why transforming with circular plasmid? This plasmid is not stable in yeast so it need recombination event to insert into the genome. If circular the recombination will occur randomly in the homologous region of plasmid and genome. Recombination will be more effective with linear plasmid and preferentially targeted to homologous regions located at the extremities... Anyway for transforming yeast with STABLE circular plasmid, 10s of ng are enough whereas if recombination is required µgs are required.
Dear Tri Minh Vo
I typically use 2-3 µg of linearized pPICZalpha-A plasmid DNA.
You must digest it first (linearize) and then purify it. Then transform your fresh electrically competent cells of Pichia strain using ~10 micro grams of linearized plasmid having your insert.
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