Laboratory: agarose gel

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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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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.
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Natural Competence import DNA Due 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
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