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Lecture7 - MCB 110L Spring 2011 Lecture 7 Qing Zhong 306...

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MCB 110L Spring 2011 Lecture 7 2/9/2011 Qing Zhong 306 Barker Hall
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Zimmerman & Trach (1991) JMB 222, 599-620. David Goodsell, http://www.scripps.edu/pub/goodsell/ E. coli contents RNA, DNA and proteins, mg/mL Exponential growth ~300 Stationary phase ~400 Plasmid DNA Isolation
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Plasmid DNA Isolation We need to separate plasmid DNA from all other cell contents Separate from proteins, RNA, membranes, chromosomal DNA Multi-step process Lyse cells (alkaline lysis in presence of RNase A) » Invert GENTLY to keep chromosomal DNA from fragmenting » Detergent and alkali lyse cells; alkali helps hydrolyse RNAs Precipitate cell debris with SDS:K+ protein precipitate and neutralize pH ( with acidic K+ acetate) » If you are careful to invert gently during lysis, chromosomal DNA will precipitate at this step caught up in debris and unable to renature » Proteins precipitate with SDS:K+. Denature residual proteins in Guanidinium HCl (recall PCR purification)
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SC - supercoiled CCC - covalently closed circular OC - open circular l - linear Different forms of plasmid DNA
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• Rate of movement is: Proportional to charge and voltage gradient » protein charge or electric field (voltage), rate Inversely proportional to size, shape, viscosity of the medium, and distance between electrodes » protein size, radius of gyration, medium viscosity, or distance between electrodes, rate General principles of electrophoresis
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