Transcript Transformation Lab

Laboratory:
Bacterial Transformation
Introduction of plasmid (pGal) DNA into
E. coli
This laboratory is
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The first part in a series of 3 experiments:
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Plasmid Transformation
Plasmid Isolation
Plasmid Mapping
Principle of transformation experiment
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Genotype determines phenotype
Your plasmid contains
Amp r
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A ampr gene
And a lac z gene
pGal
Lac Z gene
Lac Z gene
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Codes for beta-galactosidase
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Beta-galactosidase is secreted by the
transformed E. coli
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Beta-galactosidase utilizes the substrate “Xgal” to produce a blue color
Amp resistance gene
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Beta-lactamase secreted extracellularly
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Beta-lactamase inactivates ampicillin
How to transform cells
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Competent bacterial cells are required
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Introduction of plasmid DNA + bacteria
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“Heat Shock” to increase uptake of DNA
How do we know if transformation occurred?
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You must “plate” your transformed and allow
your bacteria to grow
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Identify transformed DNA by a “selectable
marker”.
Group materials
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Each group
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Plasmid (pGal) DNA
Buffer
Recovery broth
3 agar plates
3 transfer pipets or use micropipettors
2 “yellow platers”
Laboratory Protocol
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Always wear gloves
Reagents are on cart or front bench
Ice is on front bench
Divide into groups
Dispose of material in red container in front of
room
Plating of transformed bacteria
Cell spreader
Gently spread across surface
Let plate sit 10-15 min.
Cover
Incubate 37 overnight
Agar plate with drops of transformed cells
Cart and lab bench has the supplies: come get your supplies
9-10 groups!
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Each lab group should have at their bench
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1 plate labeled X-GAL
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2 plates labeled “AMP/X-GAL”
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1 microtest tube labeled “pGal DNA”
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1 microtest tube labeled “Control Buffer”
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1 microtest tube labeled “Cells for DNA”
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1 microtest tube labeled “Cells for Control”
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1 microtest tube labeled “Recovery”
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4 sterile 1 mol pipets
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1 inoculating loop
Next lab: Transformation Efficiency
is Determined
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# of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)=
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# of transformants per ug of DNA
Our experiment uses:
DNA concentration: 0.025 ug
Recovery Volume: .68 ml
Plating Volume:
0.25 ml