1. Methods of transformation
Electroporation
Electrical shock makes cell membranes permeable to DNA
Makes holes in cells
Calcium Chloride/Heat-Shock
Chemically-competent cells uptake DNA after heat shock
We will do this method

2. Bacterial Transformation
Look at figure 5.7
Competent E. coli are able to take up the plasmid
Transfer plasmid DNA into bacteria
Transfer to selective media
Grow overnight
Pick colonies – miniprep or chromatography

3. Transformation procedure
Suspend bacterial colonies in Transformation solution
Add S3 or pGLO plasmid DNA
Put cells in CaCl2 = Ca ++ binds to PO4-
Place tubes on ice
Heat-shock at 42°C and place on ice
Incubate with nutrient broth
Streak plates

4. Bacterial Transformation
Cell wall
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

5. Transformation Procedure Overview
Day 1
Day 2

6. Selection Plated transformed cells on antibiotic – Ampicillin
Only a small % of the cells will undergo transformation
Those cells that grow contain the plasmid
Remember, Arabinose causes cells to express GFP and grow green

7. Recombinant DNA = plasmid + insert
How do you know if insert is present?
Restriction digest
Blue/White selection (figure 5.10)
pJET1.2plasmid (figure 5.11)

8. Blue/White Selection (Fig. 5.10)
If fragment is not inserted, bacterial cells will be blue
If fragment is inserted and LacZ is disrupted, bacterial cells will be white
LacZ = b-galactasidase cleaves artificial substrate X gal to produce blue colonies

9. pJET1.2 plasmid (fig 5.11) Ec0471R is toxic
If ligate a fragment before Ec0471R, you will inactive the gene and bacterial cells will grow.
If there is no insert, Ec0471R will be toxic and the bacterial cells will die.

10. Transformation efficiency
Page 143 – 144
We will look back at these calculations after we perform our transformations.