Laboratory : agarose gel & transformation Lecture : reporter genes; transformation
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Transcript of Laboratory : agarose gel & transformation Lecture : reporter genes; transformation
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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
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Reporter genes monitor promoter activity or protein expression.
Promoter RFP coding sequence
Red Fluorescent Protein coding sequence fused to promoter.
Visually monitor.
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Green Fluorescent Protein fused to GALLS
Monitor expression & location
Promoter GALLS coding sequence GFP
Fusion protein
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Transformation introduces plasmid DNA into E. coli.
You will: 1) transform pKN800 DNA into E. coli, 2) select ampicillin-resistant transformants3) score colonies for luminescence.
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Natural Competence: import DNADue to growth stages; environmental signals.
gram-positive: Streptococcus, Bacillus
gram-negative: Neisseria, Haemophilus, Vibrio cholera
Chemical Competence: CaCl2; RbCl2
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa)
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Chemical transformation: ice-cold CaCl2 or RbCl2 heat shock plasmid DNA into E. coli. Electroporation: pulses of high voltage DNA into E. coli & other species.
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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.
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Linear DNA usually faster than circular:
Circular DNA 2 forms: covalently closed circular (ccc) &open circular (oc).
Closed circular DNA supercoils= twisted telephone cord.
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Small supercoiled DNA faster than same length linear.
Most supercoiled DNA slower than corresponding linear molecules.
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Break in 1 strand of circular DNA no supercoiling "relaxed" or "open" circular DNA migrates much more slowly.
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DNA stained with ethidium bromide. Ethidium bromide stacks between bases. Stained DNA + UV orange light.
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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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pKN800 A pKN800 B
10.08.0
5.0
3.54.0
3.02.5
2.0
1.5
1.0
0.75
6.0
PstI uncut PstI uncut
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