1. Laboratory: Bacterial Transformation
Introduction of plasmid (pGal) DNA into E. coli

2. This laboratory is The first part in a series of 3 experiments:
Plasmid Transformation
Plasmid Isolation
Plasmid Mapping

3. Principle of transformation experiment
Genotype determines phenotype

4. Your plasmid contains A ampr gene And a lac z gene Amp r pGal

5. Lac Z gene Codes for beta-galactosidase
Beta-galactosidase is secreted by the transformed E. coli
Beta-galactosidase utilizes the substrate “X-gal” to produce a blue color

6. Amp resistance gene Beta-lactamase secreted extracellularly
Beta-lactamase inactivates ampicillin

7. How to transform cells Competent bacterial cells are required
Introduction of plasmid DNA + bacteria
“Heat Shock” to increase uptake of DNA

8. How do we know if transformation occurred?
You must “plate” your transformed and allow your bacteria to grow
Identify transformed DNA by a “selectable marker”.

9. Group materials Each group Plasmid (pGal) DNA Buffer Recovery broth
3 agar plates
3 transfer pipets or use micropipettors
2 “yellow platers”

10. Laboratory Protocol 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

12. Plating of transformed bacteria
Cell spreader
Gently spread across surface
Let plate sit min.
Cover
Incubate 37 overnight
Agar plate with drops of transformed cells

13. Cart and lab bench has the supplies: come get your supplies 9-10 groups!
Each lab group should have at their bench
1 plate labeled X-GAL
2 plates labeled “AMP/X-GAL”
1 microtest tube labeled “pGal DNA”
1 microtest tube labeled “Control Buffer”
1 microtest tube labeled “Cells for DNA”
1 microtest tube labeled “Cells for Control”
1 microtest tube labeled “Recovery”
4 sterile 1 mol pipets
1 inoculating loop

14. Next lab: Transformation Efficiency is Determined
# of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)=
# of transformants per ug of DNA
Our experiment uses:
DNA concentration: ug
Recovery Volume: ml
Plating Volume: ml