Lecture7 - MCB 110L Spring 2011 Lecture 7 2/9/2011 Qing...

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Unformatted text preview: MCB 110L Spring 2011 Lecture 7 2/9/2011 Qing Zhong 306 Barker Hall 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 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 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+....
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Lecture7 - MCB 110L Spring 2011 Lecture 7 2/9/2011 Qing...

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