Lec7W11 - Ligation results Plasmids Ori Selectable markers...

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Ligation results Plasmids: Ori Selectable markers - Polylinker region lacZ alpha
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A B C D A = Sal 1 digested Vibrio DNA B = undigested Vibrio DNA C = Sal 1 digested pGEM D = undigested pGEM Gel from lab 6
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+ = For ligations, you took different amounts of digested Vibrio DNA (tube A) and added digested pGEM (tube C) Ligation T0 tube (no ligase) C A
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Ligation results You have two sets of tubes – both had linear pGEM and digested Vibrio DNA added to them But – The tubes labeled L1 T0 did not have ligase added – The tubes labeled L1 Tend did have ligase added to them In the tubes that ligase was added, we should have a lot of re-ligated pGEM – this will run like supercoiled pGEM on a gel with SybrSafe in it.
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L1 T0 L1 Tend L3 T0 L3 Tend L2 T0 L2 Tend Tend What ligation gels should look like today Linear pGEM Re-ligated pGEM L5 tubes: neither had ligase
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Competence and transformation Found in 1970s that treatment of E. coli cells in mid-log phase with CaCl 2 at 0-5 o C followed by heat shock allows bacteria to take up phage DNA Hypothesis is that divalent cation shields negative charge of DNA and membrane from repulsing each other, so DNA can adhere to bacterial cell membrane Then heat shock at 42 o C afterwards seems to help get DNA across membrane and into the cell
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Growth of bacteria
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http://www.dnalc.org/resources/animations/transformation2.html
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This note was uploaded on 04/10/2011 for the course BIMM 101 taught by Professor Butler during the Spring '08 term at UCSD.

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Lec7W11 - Ligation results Plasmids Ori Selectable markers...

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