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Microbio Lab 6

Plasmid Miniprep, measure of DNA, restriction digest

QuestionAnswer
name of plasmid pLL3-HRP
zeocin resistance gene allows for selection and maintenance of plasmid in bacteria and yeast
5'AOX1 sequence harbor methanol-inducible promoter of alcohol oxidase gene, turns on HRP expression in pichia pastoris (yeast) - this is off in e. coli
when will HRP be expressed if yeast is grown in methanol
restriction enzyme Mss1; linearizes plasmid
what does 7x lysis buffer made of? do? NaoH + SDS (sodium dodecyl sulfate)
Neutralization buffer made of potassium acetate and SDS traps yellow pellet while plasmid DNA reanneals in liquid
2 washes: Endo and Zyppy endotoxin removal = Endo salt removal = Zyppy
Zyppy elution plasmid released from silica column due to low salt, high pH
what is the foreign protein to be expressed? horseradish peroxidase
what does zeocin do? kills e. coli and p. pastoris cells by causing double stranded breaks in DNA
what does SDS do sodium dodecyl sulfate. denatures proteins.
what does NaOH do it denatures plasmid DNA
1.0 OD 260 = 50 micrograms/mL of DNA
A260/A280 should be greater than 1.7 to get pure DNA
Created by: syeonie
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