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. General Laboratory Practices
Wear disposable gloves, especially when handling cells, DNA, and agarose gels
Obtain reagents from front bench or cart
Keep sterilized materials clean, minimize contact with air and hands
Dispose of waste material in red biohazard bags unless otherwise instructed
Read laboratory instructions before doing the experiment

4. TRANSFORMATION Uptake of DNA from the external environment
In this experiment, we will add plasmid DNA to bacterial cells. Those that take up the plasmid will be transformed. The incoming DNA alters their genotype and observable phenotype.

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

6. How do we know if transformation occurred?
You must “plate” your transformed bacteria.
We identify transformed cells by selectable markers.
Ampicillin Resistance
Beta-galactosidase activity (causes color change)

7. Group materials Each group (4 persons) Micropipettors + Tips
Plasmid DNA
Buffer
Competent Cells
Recovery broth
3 agar plates: X-gal only (1), Amp + X-gal (2)
3 transfer pipets
1 “yellow plater”

8. Flow Chart for Transformation
Follow page 2-17 !!!!!
Flow Chart for Transformation
300 ul cells
300 ul cells
Use transfer
pipette to place
ENTIRE cell suspension
in each tube
Control
25 ul buffer
pGAL DNA
25 ul plasmid

9. Flow Chart for Transformation
Follow page 2-17 !!!!!
Flow Chart for Transformation
Incubate 10 min. on ice
Incubate 42 oC for 90 seconds
Place on ice for 1 minute
Add 0.7 ml recovery broth
Incubate at 37 oC for 30 min
Add 0.25 ml of cells to each plate
DNA
Control
Amp/X-Gal
X-Gal
Amp/Xgal

10. Plating of transformed bacteria
Cell spreader
Gently spread across surface
Let plate sit min.
Cover
Incubate at 37oC overnight
Agar plate with drops of transformed cells
Place 10 drops of 25 ul each on the plate

11. Micropipetting
Select the appropriate sized pipettor and dial in the volume needed
Use the end of the pipettor to pick up a disposable tip, use your hand to tighten the tip only at the largest end
Push the plunger to the first stop, insert the tip beneath the liquid and slowly release the plunger
Insert tip into receptacle (either against tube wall or beneath surface of liquid), push plunger to second stop to release contents
Release used tip into biohazard bag

12. Selectable Markers on the Plasmid
Protein Product:
Beta-lactamase
pUC8
Amp r
Makes bacteria
resistant to
ampicillin
Protein Product:
Beta-galactosidase
2665 bp
Lac Z gene
Cleaves sugars,
Utilizes X-gal to
produce a blue
color
Plasmid= a Circular, Independently-
replicating Piece of DNA

13. Applying Your Knowledge
Cells that take up the pUC plasmid should grow on plates containing
1. Ampicillin
2. X-gal
3. Both Ampicillin + X-gal
4. Neither Ampicillin nor X-gal

14. Applying Your Knowledge
What color will colonies of Control cells (without the pUC plasmid) be on a plate containing X-gal?
1. White
2. Blue
3. Either white or blue
4. Both white and blue
5. Neither white nor blue

15. Applying Your Knowledge
What color will colonies of Transformed cells (with the pUC plasmid) be on a plate containing X-gal?
1. White
2. Blue
3. Either white or blue
4. Both white and blue
5. Neither white nor blue

16. Next lab: Transformation Efficiency is Determined
Number of Transformants amount of DNA used (ug)
Final Recovery Volume volume plated (ml) X =
Number of Transformants per ug
Our experiment uses:
DNA concentration: ug
Recovery Volume: ml
Plating Volume: ml