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

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

3. Transformation A process of plasmid DNA uptake
In our experiment the plasmid is: extrachromosomal

4. Transformation experiment illustrates:
Genotype determines phenotype

5. Plasmid DNA
How will the phenotype of the E. coli be changed?

6. Plasmids have selectable markers to detect change:
Color alteration of colonies
Antibiotic resistance

7. Let’s look more closely at “our” plasmid
Amp r
pGal
Lac Z gene

8. What are characteristics of the lac Z gene?

9. 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

10. What are characteristics of ampr gene?

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

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

13. Bacterial Tranformation
Protocol

14. Experimental overview:
Please refer to your lab manual.

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

16. 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

17. SUMMARY This is in your lab manual! Treatment Control Amp/X-Gal X-Gal
Incubate 10 min. on ice
Incubate 42 C for 90 seconds
Place on ice for 1 minute
Add 0.75 ml recovery broth to control and treatment tubes
Incubate at 37 C min
streak 10 drops of cells evenly
Treatment
Control
Amp/X-Gal
X-Gal
Amp/Xgal

18. 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