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Laboratory: agarose gel & transformation Lecture: reporter genes; transformation In-Class Writing: restriction maps (page 24) Hand In: nothing Read: Appl.

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Presentation on theme: "Laboratory: agarose gel & transformation Lecture: reporter genes; transformation In-Class Writing: restriction maps (page 24) Hand In: nothing Read: Appl."— Presentation transcript:

1 Laboratory: agarose gel & transformation Lecture: reporter genes; transformation In-Class Writing: restriction maps (page 24) Hand In: nothing Read: Appl. Environ. Microbiol. 63: 4920-8, 1997 Due Next Class: report 1 draft

2 Reporter genes monitor promoter activity or protein expression. Promoter RFP coding sequence Red Fluorescent Protein coding sequence fused to promoter. Visually monitor.

3 Green Fluorescent Protein fused to GALLS Monitor expression & location Promoter GALLS coding sequence GFP Fusion protein

4 Transformation introduces plasmid DNA into E. coli. You will: 1) transform pKN800 DNA into E. coli, 2) select ampicillin-resistant transformants 3) score colonies for luminescence.

5 Natural Competence: import DNA Due to growth stages; environmental signals. gram-positive: Streptococcus, Bacillus gram-negative: Neisseria, Haemophilus, Vibrio cholera Chemical Competence: CaCl 2 ; RbCl 2 Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa)

6 Chemical transformation: ice-cold CaCl 2 or RbCl 2  heat shock  plasmid DNA into E. coli. Electroporation: pulses of high voltage  DNA into E. coli & other species.

7 Agarose gel electrophoresis separates DNA by size & structure. DNA  negative charge  migrates from cathode (negative, black lead) to anode (positive, red lead). Agarose = molecular sieve Retards long DNA more than short DNA.

8 Linear DNA usually faster than circular: Circular DNA  2 forms: covalently closed circular (ccc) & open circular (oc). Closed circular DNA  supercoils = twisted telephone cord.

9 Small supercoiled DNA  faster than same length linear. Most supercoiled DNA slower than corresponding linear molecules.

10 Break in 1 strand of circular DNA  no supercoiling  "relaxed" or "open" circular DNA  migrates much more slowly.

11 DNA stained with ethidium bromide. Ethidium bromide stacks between bases. Stained DNA + UV  orange light.

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13 Estimate size of restriction fragments. Compare mobility to size standards. Make standard curve: log [size] = Y-axis migration distance = X-axis for standard bands. Measure migration of restriction fragments; interpolate from standard curve to estimate sizes.

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16 pKN800 ApKN800 B 10.0 8.0 5.0 3.5 4.0 3.0 2.5 2.0 1.5 1.0 0.75 6.0 PstI uncut

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